Systems and methods to repair tissue defects

ABSTRACT

Methods of bioprinting a bio-ink construct on an internal tissue defect or a chondral defect during a minimally invasive surgery on an individual in need thereof are provided, comprising: visualizing the defect; positioning a bioprinter comprising a printhead within proximity of or in contact with the defect; and ejecting a bio-ink from the printhead onto the defect to form a bio-ink layer, thereby generating a bio-ink construct. Further provided are systems for bioprinting a bio-ink construct on an internal tissue defect during a minimally invasive surgery on an individual in need thereof, comprising a control system, an endoscope, and a bioprinter comprising a printhead.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional Application No.62/341,914, filed on May 26, 2016, which is incorporated herein byreference in its entirety.

SUMMARY OF THE INVENTION

Disclosed herein, in certain embodiments, are methods of bioprinting abio-ink construct on an internal tissue defect during a minimallyinvasive surgery on an individual in need thereof, comprising: (a)visualizing the internal tissue defect; (b) positioning a bioprintercomprising a printhead within proximity of or in contact with theinternal tissue defect; and (c) ejecting a bio-ink from the printheadonto the internal tissue defect to form a bio-ink layer, therebygenerating a bio-ink construct. In some embodiments, the bio-inkconstruct comprises a plurality of bio-ink layers. In some embodiments,the bio-ink construct is a live tissue. In some embodiments, theprinthead comprises a needle, an extended cylinder, a fluid line, aprint nozzle, or a plurality of print nozzles. In some embodiments, eachprint nozzle of the plurality of print nozzles is independentlycontrolled and actuated. In some embodiments, each print nozzle of theplurality of print nozzles is actuated to eject an individual droplet ofthe bio-ink. In some embodiments, the plurality of print nozzles ejectsthe individual droplet simultaneously. In some embodiments, theplurality of print nozzles ejects the individual droplet in a specifiedsequence. In some embodiments, the printhead ejects the bio-inkcontinuously. In some embodiments, the bio-ink comprises a plurality ofcells, a component of extracellular matrix, a synthetic polymer, anatural polymer, a cross-linking agent, a photoinitiator, or acombination thereof. In some embodiments, the plurality of cellscomprises cells selected from chondrocytes, connective tissuefibroblasts, tendon fibroblasts, bone marrow reticular tissuefibroblasts, non-epithelial fibroblasts, pericytes, osteoprogenitorcells, osteoblasts, osteoclasts, keratinocytes, hair root cells, hairshaft cells, hair matrix cells, exocrine secretory epithelial cells,hormone secreting cells, epithelial cells, neural or sensory cells,photoreceptor cells, muscle cells, extracellular matrix cells, bloodcells, cardiovascular cells, endothelial cells, kidney cells, hepaticcells, pancreatic cells, immune cells, stem cells, germ cells, nursecells, interstitial cells, stellate cells and progenitors thereof. Insome embodiments, the plurality of cells comprises cells selected fromchondrocytes, connective tissue fibroblasts, tendon fibroblasts, bonemarrow reticular tissue fibroblasts, non-epithelial fibroblasts,pericytes, osteoprogenitor cells, osteoblasts, osteoclasts, andprogenitors thereof. In some embodiments, the plurality of cellscomprises chondrocytes. In some embodiments, the component ofextracellular matrix comprises collagen, elastin, fibrillin,fibronectin, laminin, fibrinogen, tenascin, thrombospondin, integrin,hyaluronic acid, heparin, heparin sulfate, chondroitin sulfate, keratinsulfate, dermatan sulfate, or a combination thereof. In someembodiments, the synthetic polymer is polyethylene glycol (PEG), a PEGmacromere, polyethylene glycol methacrylate (PEGMA), polyethylenedimethacrylate (PEDGMA), poly(hydroxyethyl methacrylate) (PHEMA),polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), carboxymethylcellulose (CMC), polyimide (PI), polyacrylate (PAA), polyurethane (PU),PEG-lactide, PEG-glycolide, or a combination thereof. In someembodiments, the natural polymer is alginate, cellulose, gelatin,pectin, agarose, chitosan, or a combination thereof. In someembodiments, the cross-linking agent comprises calcium chloride, calciumsulfate, calcium carbonate, calcium (Ca2+), magnesium (Mg2+),glutaraldehyde, genipin, nordihydroguaiaretic acid, tannin acid,procyanidins, glycosaminoglycan (GAG),1-ethyl-3-3-dimethylaminopropylcarbodiimide hydrochloride (EDC), divinylbenzene (DVB), ethylene glycol dimethacrylate (EGDMA), tetraethyleneglycol diacrylate (TEGDA), polyethylene glycol diacrylate (PEGDA), or acombination thereof. In some embodiments, the method comprisespolymerizing the bio-ink. In some embodiments, polymerizing the bio-inkcomprises cross-linking the bio-ink. In some embodiments, cross-linkingthe bio-ink comprises delivering the cross-linking agent by theprinthead to the bio-ink. In some embodiments, cross-linking the bio-inkcomprises applying UV light from a light source to the bio-ink. In someembodiments, cross-linking the bio-ink comprises applying heat to thebio-ink. In some embodiments, the bioprinter comprises a secondprinthead. In some embodiments, the method comprises positioning asecond bioprinter comprising a printhead within proximity of or incontact with the internal tissue defect. In some embodiments, the methodcomprises ejecting a second bio-ink from the printhead of the secondbioprinter onto the internal tissue defect to form a second bio-inklayer. In some embodiments, the method comprises controlling thebioprinter with a control system. In some embodiments, the controlsystem comprises a computer system. In some embodiments, the controlsystem comprises a robotic arm operatively connected to the computersystem. In some embodiments, the robotic arm is coupled to a body partof the individual. In some embodiments, the robotic arm positions thebioprinter. In some embodiments, the bioprinter is moved along an X, Y,or Z axis, or a combination thereof. In some embodiments, the bioprinteris rotated around the X, Y, or Z axis, or a combination thereof. In someembodiments, the control system controls a bio-ink printing parameter.In some embodiments, the bio-ink printing parameter comprisestemperature, backpressure, drops per nozzle, frequency of drop rate,number of nozzles in use, firing energy, resolution, viscosity, cellconcentration, physiological temperature, speed of printing, or acombination thereof. In some embodiments, visualizing the internaltissue defect occurs before, during, or after ejecting the bio-ink. Insome embodiments, visualizing the internal tissue defect comprisesimaging the internal tissue defect. In some embodiments, the methodcomprises positioning an endoscope within proximity of the internaltissue defect. In some embodiments, the endoscope visualizes theinternal tissue defect. In some embodiments, the internal tissue defectis selected from a damaged tissue, eroded tissue, diseased tissue ordegenerated tissue. In some embodiments, the internal tissue defect isin an internal tissue selected from bone, muscle, nerves, brain, eye,pancreas, spleen, cartilage, thyroid, adipose, sinus, esophagus, kidney,heart, lung, intestine, stomach, colon, rectum, breast, ovary, uterus,cervix, prostate, bladder or liver. In some embodiments, the internaltissue defect is selected from a vascular defect, a chondral defect, amuscular defect, an intestinal defect, a neuronal defect, a reproductivedefect, a pancreatic defect, or an ocular defect. In some embodiments,the internal tissue defect comprises a chondral defect. In someembodiments, the chondral defect is in a joint selected from a kneejoint, a hip joint, an elbow joint, a shoulder joint, a wrist joint, aspine joint, a finger joint, an ankle joint, or a foot joint. In someembodiments, the chondral defect is in a knee joint. In someembodiments, the chondral defect is an osteochondral defect.

Disclosed herein, in certain embodiments, are methods of bioprinting abio-ink construct on a chondral defect during a minimally invasivesurgery on an individual in need thereof comprising: (a) visualizing thechondral defect; (b) positioning a bioprinter comprising a printheadwithin proximity of or in contact with the chondral defect; and (c)ejecting a bio-ink from the printhead onto the chondral defect to form abio-ink layer, thereby generating a bio-ink construct. In someembodiments, the bio-ink construct comprises a plurality of bio-inklayers. In some embodiments, the bio-ink construct is a live tissue. Insome embodiments, the printhead comprises a needle, an extendedcylinder, a fluid line, a print nozzle, or a plurality of print nozzles.In some embodiments, each print nozzle of the plurality of print nozzlesis independently controlled and actuated. In some embodiments, eachprint nozzle of the plurality of print nozzles is actuated to eject anindividual droplet of the bio-ink. In some embodiments, the plurality ofprint nozzles ejects the individual droplet simultaneously. In someembodiments, the plurality of print nozzles ejects the individualdroplet in a specified sequence. In some embodiments, the printheadejects the bio-ink continuously. In some embodiments, the bio-inkcomprises a plurality of cells, a component of extracellular matrix, asynthetic polymer, a natural polymer, a cross-linking agent, aphotoinitiator, or a combination thereof. In some embodiments, theplurality of cells comprises cells selected from chondrocytes,connective tissue fibroblasts, tendon fibroblasts, bone marrow reticulartissue fibroblasts, non-epithelial fibroblasts, pericytes,osteoprogenitor cells, osteoblasts, osteoclasts, and progenitorsthereof. In some embodiments, the plurality of cells compriseschondrocytes. In some embodiments, the component of extracellular matrixcomprises collagen, elastin, fibrillin, fibronectin, laminin,fibrinogen, tenascin, thrombospondin, integrin, hyaluronic acid,heparin, heparin sulfate, chondroitin sulfate, keratin sulfate, dermatansulfate, or a combination thereof. In some embodiments, the syntheticpolymer is polyethylene glycol (PEG), a PEG macromere, polyethyleneglycol methacrylate (PEGMA), polyethylene dimethacrylate (PEDGMA),poly(hydroxyethyl methacrylate) (PHEMA), polyvinyl alcohol (PVA),polyvinylpyrrolidone (PVP), carboxymethyl cellulose (CMC), polyimide(PI), polyacrylate (PAA), polyurethane (PU), PEG-lactide, PEG-glycolide,or a combination thereof. In some embodiments, the natural polymer isalginate, cellulose, gelatin, pectin, agarose, chitosan, or acombination thereof. In some embodiments, the cross-linking agentcomprises calcium chloride, calcium sulfate, calcium carbonate, calcium(Ca2+), magnesium (Mg2+), glutaraldehyde, genipin, nordihydroguaiareticacid, tannin acid, procyanidins, glycosaminoglycan (GAG),1-ethyl-3-3-dimethylaminopropylcarbodiimide hydrochloride (EDC), divinylbenzene (DVB), ethylene glycol dimethacrylate (EGDMA), tetraethyleneglycol diacrylate (TEGDA), polyethylene glycol diacrylate (PEGDA), or acombination thereof. In some embodiments, the method comprisespolymerizing the bio-ink. In some embodiments, polymerizing the bio-inkcomprises cross-linking the bio-ink. In some embodiments, cross-linkingthe bio-ink comprises delivering the cross-linking agent by theprinthead to the bio-ink. In some embodiments, cross-linking the bio-inkcomprises applying UV light from a light source to the bio-ink. In someembodiments, cross-linking the bio-ink comprises applying heat to thebio-ink. In some embodiments, the bioprinter comprises a secondprinthead. In some embodiments, the method comprises positioning asecond bioprinter comprising a printhead within proximity of or incontact with the chondral defect. In some embodiments, the methodcomprises ejecting a second bio-ink from the printhead of the secondbioprinter onto the chondral defect to form a second bio-ink layer. Insome embodiments, the method comprises controlling the bioprinter with acontrol system. In some embodiments, the control system comprises acomputer system. In some embodiments, the control system comprises arobotic arm operatively connected to the computer system. In someembodiments, the robotic arm is coupled to a body part of theindividual. In some embodiments, the robotic arm positions thebioprinter. In some embodiments, the bioprinter is moved along an X, Y,or Z axis, or a combination thereof. In some embodiments, the bioprinteris rotated around the X, Y, or Z axis, or a combination thereof. In someembodiments, the control system controls a bio-ink printing parameter.In some embodiments, the bio-ink printing parameter comprisestemperature, backpressure, drops per nozzle, frequency of drop rate,number of nozzles in use, firing energy, resolution, viscosity, cellconcentration, physiological temperature, speed of printing, or acombination thereof. In some embodiments, visualizing the chondraldefect occurs before, during, or after ejecting the bio-ink. In someembodiments, visualizing the chondral defect comprises imaging thechondral defect. In some embodiments, the method comprises positioningan endoscope within proximity of the chondral defect. In someembodiments, the endoscope visualizes the chondral defect. In someembodiments, the chondral defect is selected from a damaged tissue,eroded tissue, diseased tissue or degenerated tissue. In someembodiments, the chondral defect is in a joint selected from a kneejoint, a hip joint, an elbow joint, a shoulder joint, a wrist joint, aspine joint, a finger joint, an ankle joint, or a foot joint. In someembodiments, the chondral defect is in a knee joint. In someembodiments, the chondral defect is an osteochondral defect.

Disclosed herein, in certain embodiments, are biological compositiondelivery systems comprising an endoscope, at least one bioprintercomprising at least one printhead, and a control system that controlsthe at least one bioprinter; wherein the biological composition is abio-ink comprising a plurality of cells. In some embodiments, theplurality of cells is a plurality of autologous cells, allogeneic cells,or a combination thereof. In some embodiments, the plurality of cells isa plurality of chondrogenic precursors. In some embodiments, theplurality of cells is a plurality of pancreatic cells. In someembodiments, the plurality of cells is a plurality of hepatic cells. Insome embodiments, the plurality of cells is a plurality of neural cells.In some embodiments, the plurality of cells is a plurality of retinalcells. In some embodiments, the plurality of cells is a plurality ofimmunologic cells. In some embodiments, the plurality of cells is aplurality of renal cells. In some embodiments, the plurality of cells isa plurality of hematopoietic cells. In some embodiments, the pluralityof cells is a plurality of adipose cells. In some embodiments, theplurality of cells is a plurality of fibroblastic cells. In someembodiments, the plurality of cells is a plurality of osteoblasticcells. In some embodiments, the plurality of cells is a plurality ofmuscle cells. In some embodiments, the plurality of cells is a pluralityof epithelial cells. In some embodiments, the plurality of cells is aplurality of endothelial cells. In some embodiments, the biologicalcomposition delivery system comprise a three dimensional scanner. Insome embodiments, the three dimensional scanner is configured to createa point cloud of an internal tissue defect, a bio-ink construct, or acombination thereof. In some embodiments, the point cloud is used todesign the bio-ink construct that complements the shape of the internaltissue defect of a patient. In some embodiments, the at least oneprinthead comprises a needle, a print nozzle, or a combination thereof.In some embodiments, the biological composition delivery systemscomprise a first bioprinter comprising a first printhead and a secondbioprinter comprising a second printhead; wherein the first printheadejects a first bio-ink and the second printhead ejects a second bio-ink.In some embodiments, the system is portable. In some embodiments, thecontrol system comprises a robotic arm operatively connected to acomputer system. In some embodiments, the robotic arm: 1) positions thebioprinter within proximity of or in contact with an internal tissuedefect, and 2) has six degrees of freedom. In some embodiments, thecontrol system comprises a second robotic arm operatively connected to acomputer system, wherein the second robotic arm has six degrees offreedom. In some embodiments, the endoscope is configured to provide animage of the internal tissue defect; wherein the image is used toprovide feedback regarding the structure of a bio-ink construct during abio-printing process in real time. In some embodiments, the plurality ofcells is selected from chondrocytes, mesenchymal stem cells (MSCs),MSC-like cells, human embryonic stem cells (hESCs), induced pluripotentstem cells (iPSCs), or a combination thereof. In some embodiments, theplurality of cells is a plurality of chondrocytes. In some embodiments,the plurality of cells is a plurality of mesenchymal stem cells (MSCs).In some embodiments, the plurality of cells is a plurality of humanembryonic stem cells (hESCs). In some embodiments, the plurality ofcells is a plurality of induced pluripotent stem cells (iPSCs). In someembodiments, the bio-ink comprises a component of extracellular matrix.In some embodiments, the bio-ink comprises polylactic acid,methacrylated collagen, or a combination thereof. In some embodiments,the bio-ink comprises collagen. In some embodiments, the bio-inkcomprises a cross-linking agent, a photoinitiator, or a combinationthereof.

BRIEF DESCRIPTION OF THE DRAWINGS

A better understanding of the features and advantages of the presentinvention will be obtained by reference to the following detaileddescription that sets forth illustrative embodiments, in which theprinciples of the invention are utilized, and the accompanying drawingsof which:

FIGS. 1A-1F illustrate different heights and angles of the printnozzles. FIG. 1A illustrates a printhead with a linear array ofconverging print nozzles. FIG. 1B illustrates a printhead with a lineararray of diverging print nozzles. FIG. 1C illustrates a printhead with alinear array of print nozzles, wherein the levels of the individualprint nozzles are not all in the same plane. Shown here are printnozzles that are deeper in the center. FIG. 1D illustrates a printheadwith a two dimensional array of converging print nozzles. FIG. 1Eillustrates printhead with a two dimensional array of diverging printnozzles. FIG. 1F illustrates a two dimensional array of print nozzles,wherein the level of individual print nozzles are not all in the sameplane.

FIGS. 2A-2E illustrate different configures of print nozzles on aprinthead. FIG. 2A illustrates print nozzles evenly distributed in arectangular shape. FIG. 2B illustrates print nozzles distributed withone or more masks (an empty space devoid of print nozzles). FIG. 2Cillustrates print nozzles distributed in a star shape. FIG. 2Dillustrates print nozzles distributed in an elliptical shape. FIG. 2Eillustrates individual print nozzles of different sizes and diameters,distributed in various locations, as well as each print nozzlebioprinting a different bio-ink, different cell type, differentmolecules, bioactive factors, matrix components, and/or pharmacologicagent.

FIG. 3 illustrates a bioprinter which ejects bio-ink via syringeextrusion.

FIG. 4 illustrates a bioprinter which ejects bio-ink via diaphragm basedjetting.

FIGS. 5A-5B illustrate a bioprinter which uses ink-jet based printing.FIG. 5A illustrates a bioprinter which ejects bio-ink via ink-jet basedprinting, through a single nozzle. FIG. 5B illustrates a bioprinterwhich ejects bio-ink via ink-jet based printing through a plurality ofnozzles.

FIG. 6 exemplifies repair of an articular lesion in the knee using twobioprinters to print bio-ink, controlled by two robotic arms, and anendoscope to visualize the internal tissue defect.

DETAILED DESCRIPTION OF THE INVENTION

Bioprinting is the process of generating spatially-controlled cellpatterns using three dimensional (3D) printing technologies, where cellfunction and viability are preserved within the printed construct.Bio-printing typically involves dispensing cells onto a biocompatiblescaffold using a successive layer-by-layer approach to generatetissue-like three dimensional structures.

Surgical procedures can be performed with the systems disclosed hereinin a minimally invasive manner. The benefits of minimally invasivesurgery include less patient trauma, less blood loss, and fasterrecovery times when compared to traditional, open incision surgery.Furthermore, advantages of printing directly onto an internal tissuedefect, as provided by the methods and systems disclosed herein,include, but are not limited to: i) eliminating the need for priormanufacturing, storage, or transportation; ii) providing the ability tocustomize the engineered tissue to perfectly fit defects of any shape orsize; iii) eliminating the need to reconstruct, modify, or enlarge thedefect to match the pre-engineered shape; iv) the ability to vary thetype or amount of tissue being generated during surgery; v) the abilityto combine artificial and natural scaffolds as well as living cells; andvi) enabling direct integration of the newly printed tissue into thehost tissue.

Certain Terminology

The terminology used herein is for the purpose of describing particularcases only and is not intended to be limiting. As used herein, thesingular forms “a”, “an” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise.Furthermore, to the extent that the terms “including”, “includes”,“having”, “has”, “with”, or variants thereof are used in either thedetailed description and/or the claims, such terms are intended to beinclusive in a manner similar to the term “comprising.”

As used herein, the term “about” or “approximately” means within anacceptable error range for the particular value as determined by one ofordinary skill in the art, which will depend in part on how the value ismeasured or determined, e.g., the limitations of the measurement system.In certain embodiments, the term “about” or “approximately” means within1, 2, 3, or 4 standard deviations. In certain embodiments, the term“about” or “approximately” means within 30%, 25%, 20%, 15%, 10%, 9%, 8%,7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, or 0.05% of a given value orrange. In certain embodiments, the term “about” or “approximately” meanswithin 20.0 degrees, 15.0 degrees, 10.0 degrees, 9.0 degrees, 8.0degrees, 7.0 degrees, 6.0 degrees, 5.0 degrees, 4.0 degrees, 3.0degrees, 2.0 degrees, 1.0 degrees, 0.9 degrees, 0.8 degrees, 0.7degrees, 0.6 degrees, 0.5 degrees, 0.4 degrees, 0.3 degrees, 0.2degrees, 0.1 degrees, 0.09 degrees. 0.08 degrees, 0.07 degrees, 0.06degrees, 0.05 degrees, 0.04 degrees, 0.03 degrees, 0.02 degrees or 0.01degrees of a given value or range.

As used herein, the terms “individual,” “patient,” or “subject” are usedinterchangeably. None of the terms require or are limited to situationcharacterized by the supervision (e.g. constant or intermittent) of ahealth care worker (e.g. a doctor, a registered nurse, a nursepractitioner, a physician’s assistant, an orderly, or a hospice worker).

As used herein, the terms “user,” “health care provider,” or “surgeon”are used interchangeably and refer to the person or persons who will beoperating the bioprinter and/or the bioprinting system.

As used herein, the terms “treating” or “treatment” of a state, disorderor condition (e.g., cancer) includes: (1) preventing or delaying theappearance of clinical or sub-clinical symptoms of the disorderdeveloping in a human that is afflicted with or pre-disposed to thedisorder but does not yet experience or display clinical or subclinicalsymptoms of the disorder; and/or (2) inhibiting the disorder, includingarresting, reducing or delaying the clinical manifestation of thedisorder or at least one clinical or sub-clinical symptom thereof;and/or (3) relieving the disorder, e.g., causing regression of thedisorder or at least one of its clinical or sub-clinical symptoms;and/or (4) causing a decrease in the severity of one or more symptoms ofthe disorder. The benefit to a subject to be treated is eitherstatistically significant or at least perceptible to the patient or tothe physician.

As used herein, a “bio-ink” refers to a composition suitable forbioprinting comprising a biopolymer and/or a plurality of cells. In someembodiments, bio-ink comprises cell solutions, cell aggregates,cell-comprising gels, proteins, multicellular bodies, or tissues.

As used herein, “chondrocytes” includes chondrocytes, articularchondrocytes, fibrochondrocytes, chondroblasts, chondrocyte precursors,chondrocyte progenitors, mesenchymal stem cells, osteoblasts, immaturechondrocytes, cartilage cells, chondrogenic cells, osteogenic cells,osteoprogenitor cells, osteochondroprogenitor cells, connective tissuefibroblasts, tendon fibroblasts, and cells that support the growth ordifferentiation of such cells.

As used herein, “polymerization” refers to both the process of forming apolymer chain and the process of forming networks of polymers.Polymerization includes cross-linking of polymers, including covalentand ionic cross-linking. In some embodiments, polymerization includesgelatinization, or gelling, of the bio-ink.

As used herein, the terms “chondrogenic precursor,”“chondro-progenitor,” “chondrocyte progenitor,” “chondrocyte precursor”are used interchangeably.

As used herein, the term “embryonic stem cells” (ESCs) refers topluripotent stem cells that are derived from a blastocyst beforesubstantial differentiation of the cells into the three germ layers.ESCs include any commercially available or well established ESC cellline such as, by way of non-limiting example, H9, H1, H7, and SA002.

As used herein, the term “induced pluripotent stem cells” or “iPSCs”refers to somatic cells that have been reprogrammed into a pluripotentstate resembling that of embryonic stem cells. Included in thedefinition of iPSCs are iPSCs of various types including human iPSCs andnon-human iPSCs, such as iPSCs derived from somatic cells that areprimate somatic cells or murine somatic cells.

As used herein, the term “allogeneic” means the plurality of cells areobtained from a genetically non-identical donor. For example, allogeneiccells are extracted from a donor and returned back to a different,genetically non-identical recipient.

As used herein, the term “autologous” means the plurality of cells areobtained from a genetically identical donor. For example, autologouscells are extracted from a patient and returned back to the same,genetically identical patient.

Methods of Bioprinting

Disclosed herein, in certain embodiments, are methods of bioprinting abio-ink construct on an internal tissue defect during a minimallyinvasive surgery on an individual in need thereof, comprising:visualizing an internal tissue defect; positioning a bioprinter withinproximity of or in contact with the internal tissue defect; and ejectinga bio-ink from the bioprinter onto the internal tissue defect to form abio-ink layer, thereby generating a bio-ink construct.

Disclosed herein, in certain embodiments, are methods of bioprinting abio-ink construct on a chondral defect during a minimally invasivesurgery on an individual in need thereof, comprising: visualizing thechondral defect; positioning a bioprinter within proximity of or incontact with the chondral defect; and ejecting a bio-ink from thebioprinter onto the chondral defect to form a bio-ink layer, therebygenerating a bio-ink construct.

In some embodiments, the methods of bioprinting comprise polymerizingthe bio-ink. In some embodiments, polymerizing the bio-ink comprisesapplying a specified temperature or chemical to the bio-ink. In someembodiments, the method comprises polymerizing the bio-ink as it isprinted on the substrate. In some embodiments, the method comprisesphotopolymerizing the bio-ink as it is printed on the substrate. In someembodiments, the method comprises polymerizing the bio-ink after it isprinted on the substrate. In some embodiments, the method comprisesphotopolymerizing the bio-ink after it is printed on the substrate.

In some embodiments, the methods of bioprinting comprise gelling thebio-ink. In some embodiments, gelling the bio-ink comprises applying aspecified temperature or chemical to the bio-ink. In some embodiments,the chemical is a cross-linking agent. In some embodiments, the bio-inkundergoes gelatinization. In some embodiments, gelatinization is inducedby a change pH, a change in temperature, coulombic interactions,covalent bonding, non-covalent interactions, or polymerization.

In some embodiments, polymerizing the bio-ink comprises cross-linkingthe polymers in the bio-ink. In some embodiments, cross-linking thepolymers in the bio-ink comprises chemical cross-linking. In someembodiments, cross-linking the polymers in the bio-ink occurs after thebio-ink is printed. In some embodiments, cross-linking the polymers inthe bio-ink and printing occur simultaneously. In some embodiments,cross-linking the polymers in the bio-ink comprises cross-linking with afree radical initiator. In some embodiments, cross-linking the polymersin the bio-ink comprises crosslinking with thiol or amine moieties. Insome embodiments, cross-linking the polymers in the bio-ink comprisesdelivering a cross-linking agent to the bio-ink. In some embodiments,the cross-linking agent comprises calcium (Ca²⁺), magnesium (Mg²⁺),calcium chloride, calcium sulfate, calcium carbonate, glutaraldehyde,genipin, nordihydroguaiaretic acid, tannin acid, procyanidin,1-ethyl-3-3-dimethylaminopropylcarbodiimide hydrochloride (EDC), divinylbenzene (DVB), ethylene glycol dimethacrylate (EGDMA), tetraethyleneglycol diacrylate (TEGDA), polyethylene glycol diacrylate (PEGDA), or acombination thereof. In some embodiments, the cross-linking agent isdelivered to the bio-ink by the bioprinter.

In some embodiments, the bio-ink turns into a solid during the printingprocess. In some embodiments, the method comprises polymerization ordegradation of the bio-ink by exposure to electromagnetic radiation. Insome embodiments the electromagnetic radiation comprises an electronbeam, gamma-radiation, or UV radiation. In some embodiments, the methodcomprises polymerization or degradation of the bio-ink by exposure tolight. In some embodiments, light is used to partially degrade abioprinted tissue. In some embodiments, time, wavelength, and lightintensity of light exposure are varied. In some embodiments, degradationor polymerization are paused by shuttering the light. In someembodiments, the gel continues polymerizing or degrading once lightexposure resumes. In some embodiments, the method comprises adding aphotoinitiator to the bio-ink. In some embodiments, any suitablephotoinitiator is used. In some embodiments, the photoinitiator is aphotoinitiator for UV curing. In some embodiments a type Iphotoinitiator or a type II photoinitiator is used. Examples ofphotoinitiators include, but are not limited to, Irgacure® 2959,2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone,(2-hydroxy-1-[4-(2-hydroxyethoxy) phenyl]-2-methyl-1-propanone ; lithiumphenyl-2,4,6-trimethylbenzoylphosphinate (LAP);(2,2′-azobis[2-methyl-N-(2-hydroxyethyl)propionamide]; 2-isocyanotoethylmethacrylate; benzoyl benzylamine; camphorquinone; thiol-norbornene(thiol-ene); riboflavin; lucirin-TPO; Rose Bengal/furfuryl; ethyl eosin;methacrylic anhydride; 2,2-dimethoxy-2-phenylacetophenone; and Eosin Y.In some embodiments, the photoinitiator is added at a finalconcentration of about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.1%, 0.15%,0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% orabout 1% w/v gel. In some embodiments, the photoinitiator is added at afinal concentration of about 0.05% w/v gel. In some embodiments, themethod comprises removing bio-ink components (e.g. non-cellularcomponents, non-ECM components) after bioprinting by physical, chemical,or enzymatic means. In some embodiments, the bio-ink components areremoved by degradation of the bio-ink components.

In some embodiments, the method comprises bioprinting vascular cells. Insome embodiments, bioprinting vascular cells results in formation of ablood vessel or a portion thereof. In some embodiments, the method ofproducing the bio-ink construct comprises bioprinting extracellularmatrix components. In some embodiments, the method of producing thebio-ink construct comprises bioprinting vascular cells and extracellularmatrix components. In some embodiments, bioprinting vascular cells andextracellular matrix comprises bioprinting endothelial cells, smoothmuscle cells and fibrin. In some embodiments, the method comprisesprinting cartilage. In some embodiments, the method described hereincomprises printing a pancreatic tissue. In some embodiments, the methoddescribed herein comprises printing a hepatic tissue. In someembodiments, the method described herein comprises printing a renaltissue. In some embodiments, the method described herein comprisesprinting a bladder or ureteral tissue. In some embodiments, the methoddescribed herein comprises printing a lung tissue. In some embodiments,the method described herein comprises printing vascular tissue. In someembodiments, the method described herein comprises printing retinaltissue. In some embodiments, the method described herein comprisesprinting neural tissue. In some embodiments, the method described hereincomprises printing muscle tissue. In some embodiments, the methoddescribed herein comprises printing endothelial tissue. In someembodiments, the method described herein comprises printing epithelialtissue. In some embodiments, the method described herein comprisesprinting mucosal tissue. In some embodiments, the method describedherein comprises printing fibrous tissue. In some embodiments, themethod described herein comprises printing adipose tissue.

In some embodiments, the methods described herein comprise biomechanicaltesting of the bio-ink construct. In some embodiments, biomechanicaltesting tests the integrity of the bio-ink construct. In someembodiments, biomechanical testing comprises mechanical indentation,acoustic, ultrasonic analysis, or any suitable biomechanical testingtechnique.

In some embodiments, the methods described herein comprise bioprinting abio-ink construct that has a structure or shape that is specific to atissue defect of a patient. In some embodiments, the methods describedherein comprise bioprinting a bio-ink construct that has a meniscalshape. In some embodiments, the methods described herein comprisebioprinting a bio-ink construct that has a structure or shape thatcomplements the structure or shape of a tissue defect of a patient.

Visualizing the Defect

In some embodiments, the methods described herein comprise visualizingthe internal tissue defect. In some embodiments, the visualizing theinternal tissue defect comprises visualizing the internal tissue defectbefore, during, or after ejecting the bio-ink. In some embodiments,visualizing the internal tissue defect before ejecting the bio-ink isdone pre-operatively. In some embodiments, pre-operative visualizing ofthe internal tissue defect comprises x-ray, CAT/CT scan, PET scan, MRI,ultrasound, thermography, endoscopy, or radiography of the internaltissue defect. In some embodiments, visualizing the internal tissuedefect before ejecting the bio-ink is done during surgery, to generatean image of the to-be printed bio-ink construct. In some embodiments,visualizing the internal tissue defect is done in real-time duringejection of the bio-ink, in order to monitor the progress, fidelity, oraccuracy of printing.

In some embodiments, visualizing the internal tissue defect comprisesimaging the internal tissue defect before, during, or after ejecting thebio-ink. In some embodiments, imaging the internal tissue defectcomprises generating an image of the internal tissue defect with aphotograph, infrared imaging, three dimensional scanning, ultrasound,fluoroscopy, touch probing, or any suitable imagine technique.

In some embodiments, the image of the internal tissue defect is used todesign a bio-ink construct. In some embodiments, the image of theinternal tissue defect is used to determine the shape or structure ofthe bio-ink construct. In some embodiments, the image of the internaltissue defect is used to design a bio-ink construct that fits or alignswith a tissue defect of a patient. In some embodiments, the image of theinternal tissue defect is used to design a bio-ink construct that ispatient specific and matches the shape or construct of the tissue defectof the patient. In some embodiments, the image of the internal tissuedefect is used to program a control system to execute the bio-inkprinting parameter. In some embodiments, programming the control systemis done manually. In some embodiments, programming the control system isdone automatically. In some embodiments, the bio-ink printing parameteris altered. In some embodiments, the bio-ink printing parameter isaltered by a healthcare provider. In some embodiments, the bio-inkprinting parameter is altered during a surgical procedure.

In some embodiments, the methods comprise positioning an endoscopewithin proximity of the internal tissue defect. In some embodiments, theendoscope visualizes the internal tissue defect during bioprinting. Insome embodiments, the endoscope is an arthroscope, bronchoscope,colonscope, colposcope, cystoeurethroscope, cystoscope, duodensocope,enteroscope, esophagogastroduodenscope, fetoscope, gastroscope,gynoscope, hysterscope, laparoscope, laryngoscope, peritoneoscope,proctosigmoidoscope, sigmoidoscope, thoracoscope, or ureteroscope. Insome embodiments, the methods comprise positioning a second endoscope, athird endoscope, a fourth endoscope, or a fifth endoscope withinproximity of the internal tissue defect.

Positioning the Bioprinter

In some embodiments, the methods described herein comprise positioning abioprinter within proximity of or in contact with the internal tissuedefect. In some embodiments, the method comprises positioning a secondbioprinter within proximity of or in contact with the internal tissuedefect. In some embodiments, the bioprinter is positioned withinproximity of or in contact with the internal tissue defect by insertingthe bioprinter into the patient through an incision in the skin of thepatient. In some embodiments, the printhead is positioned withinproximity of or in contact with the internal tissue defect by insertingthe printhead into the patient through an incision in the skin of thepatient. In some embodiments, the needle is positioned within proximityof or in contact with the internal tissue defect by inserting the needleinto the patient through an incision in the skin of the patient. In someembodiments, the nozzle is positioned within proximity of or in contactwith the internal tissue defect by inserting the nozzle into the patientthrough an incision in the skin of the patient. In some embodiments, aplurality of nozzles is positioned within proximity of or in contactwith the internal tissue defect by inserting the plurality of nozzlesinto the patient through an incision in the skin of the patient.

Ejecting the Bio-Ink

In some embodiments, the methods described herein comprise bioprinting abio-ink construct comprising a bio-ink composition. In some embodiments,the methods described herein comprise ejecting a bio-ink. In someembodiments, the printhead ejects bio-ink using an extrusion printingsystem. In some embodiments, the printhead ejects bio-ink using adroplet-based printing system. In some embodiments, the printhead ejectsbio-ink using an ink-jet printing system.

In some embodiments, the extrusion printing system comprises applyingforce, heat, or a combination thereof to eject the bio-ink. In someembodiments, the force is mechanical, pneumatic, or hydraulic force. Insome embodiments, the extrusion printing system is a syringe (FIG. 3 ).In some embodiments, the syringe comprises a needle. In someembodiments, the extrusion printing system ejects the bio-inkcontinuously. In some embodiments, the extrusion printing system ejectsthe bio-ink continuously when the force or heat is applied. In someembodiments, the extrusion printing system ejects the bio-inkintermittently.

Ink-jet printing is a printing technique that reproduces digital patterninformation onto a substrate with ink drops. In some embodiments, theink-jet printing system is a thermal ink-jet system. In someembodiments, the ink jet printing system is a piezoelectric ink jetsystem. In some embodiments, the ink jet printing system uses mechanicalvibration. In some embodiments, the extrusion printing system is adiaphragm-based jetting implement (FIG. 4 ). In some embodiments, thebioprinter of FIG. 4 is connected to an air supply source (not shown),which supplies pressurized air to the bioprinter. In some embodiments,the pressurized air directs a bio-ink from a reservoir containing thebio-ink to a nozzle, thereby creating a continuous stream of bio-ink. Insome embodiments, a mechanical vibration interrupts the continuousstream of bio-ink and breaks up the stream of bio-ink into individualbio-ink droplets.

In some embodiments, the inkjet printing system comprises a heatingelement in each print nozzle. In some embodiments, the heating elementraises the local print nozzle temperature to about 100° C., about 150°C., about 200° C., about 250° C., about 260° C., about 270° C., about280° C., about 285° C., about 290° C., about 295° C., about 298° C.,about 300° C., about 302° C., about 305° C., about 310° C., about 315°C., about 320° C., about 325° C., about 350° C., about 375° C., or about400° C. In some embodiments, the heating element raises the local nozzletemperature to about 300° C. In some embodiments the heating elementraises the temperature of the plurality of cells in the bio-ink about 1°C., about 2° C., about 3° C., about 4° C., about 5° C., about 6° C.,about 7° C., about 8° C., about 9° C., about 10° C., about 11° C., about12° C., about 13° C., about 14° C. or about 15° C. In some embodiments,the temperature of the plurality of cells in the bio-ink is raised forless than about 1 µsec, about 2 µsec, about 3 µsec, about 4 µsec, about5 µsec, about 6 µsec, about 7 µsec, about 8 µsec, about 9 µsec or about10 µsec. In some embodiments, the ink-jet printing system comprises oneprint nozzle. In other embodiments, the ink-jet printing systemcomprises a plurality of print nozzles.

Bio-Ink Compositions

Disclosed herein, in certain embodiments, are bio-ink compositions. Insome embodiments, the bio-ink compositions are produced by the methodsand systems disclosed herein. Bio-ink compositions disclosed herein maybe in the form of a fluid, gel, or a construct. In some embodiments, theterm “gel” as used herein refers to a soft, solid, or solid-likecomposition that exhibits reduced or no flow when in the steady state,and it is characterized by a high viscosity. In some embodiments, thegel is a mixture. In some embodiments, the gel comprises a fluid. Insome embodiments, the gel comprises water. In some embodiments, the gelis a hydrogel, an aerogel, a nanocomposite hydrogel, a xerogel, anorganogel, a synthetic gel, a natural gel, or a combination thereof. Insome embodiments, a bio-ink composition is used in a method of treatmentdisclosed herein. In some embodiments, a bio-ink composition is used ina method of surgery of a human or an animal disclosed herein.

Bio-ink compositions disclosed herein may comprise a plurality of cells,a component of extracellular matrix, a growth factor, a therapeuticagent, a synthetic polymer, a natural polymer, a cross-linking agent, aphotoinitiator, or a combination thereof. In some embodiments, thebio-ink comprises a plurality of cells. In some embodiments, the celldensity of bio-ink is about 1 cell/pL, about 10 cells/pL, about 100cells/pL, about 1 cell/nL, about 10 cells/nL, about 100 cells/nL, about1 cell/µL, about 10 cells/µL, about 100 cells/µL, about 1000 cells/µL,about 10,000 cells cells/µL, about 100,000 cells/µL. In someembodiments, the cell density of the bio-ink is about 2×10⁶ cells/mL,about 3×10⁶ cells/mL, about 4×10⁶ cells/mL, about 5×10⁶ cells/mL, about6×10⁶ cells/mL, about 7×10⁶ cells/mL, about 8×10⁶ cells/mL, about 9×10⁶cells/mL, about 10×10⁶ cells/mL, about 15×10⁶ cells/mL, about 20×10⁶cells/mL, about 25×10⁶ cells/mL, about 30×10⁶ cells/mL, about 35×10⁶cells/mL, about 40×10⁶ cells/mL, about 45×10⁶ cells/mL, or about 50×10⁶cells/mL. In some embodiments, the plurality of cells comprises one celltype. In some embodiments, the plurality of cells comprises acombination of cell types. In some embodiments, the plurality of cellscomprises more than one cell type. In some embodiments, the plurality ofcells comprises about 2, about 3, about 4, about 5, about 6, about 7,about 8, about 9, about 10, about 12, about 14, about 16, about 18,about 20, about 25, about 30, about 35, about 40, about 45, about 50,about 55, about 60, about 65, about 70, about 75, about 80, about 85,about 90, about 95 or about 100 cell types. In some embodiments, thebio-ink comprises more than 100 cell types.

In some embodiments, the plurality of cells comprises chondrocytes,chondroprogenitor cells, keratinocytes, hair root cells, hair shaftcells, hair matrix cells, exocrine secretory epithelial cells, hormonesecreting cells, epithelial cells, neural or sensory cells,photoreceptor cells, muscle cells, extracellular matrix cells, bloodcells, cardiovascular cells, endothelial cells, vascular smooth musclecells kidney cells, pancreatic cells, immune cells, stem cells, germcells, nurse cells, interstitial cells, stellate cells liver cells,gastrointestinal cells, lung cells, tracheal cells, vascular cells,skeletal muscle cells, cardiac cells, skin cells, smooth muscle cells,connective tissue cells, corneal cells, genitourinary cells, breastcells, reproductive cells, endothelial cells, epithelial cells,fibroblasts, Schwann cells, adipose cells, bone cells, bone marrowcells, cartilage cells, pericytes, mesothelial cells, cells derived fromendocrine tissue, stromal cells, progenitor cells, lymph cells,endoderm-derived cells, ectoderm-derived cells, mesoderm-derived cells,pericytes, or progenitors thereof and/or a combination thereof In someembodiments, the plurality of cells comprises chondrocytes. In someembodiments, the plurality of cells comprises chondroblasts. In someembodiments, the bio-ink composition comprises a plurality ofchondrocytes. In some embodiments, the bio-ink composition comprises aplurality of mesenchymal stem cells. In some embodiments, the pluralityof cells comprise connective tissue fibroblasts, tendon fibroblasts,bone marrow reticular tissue fibroblasts, non-epithelial fibroblasts,pericytes, osteoprogenitor cells, osteoblasts, or osteoclasts or anycombination thereof. In some embodiments, the plurality of cellscomprises articular chondrocytes. In some embodiments, the plurality ofcells is selected from stem cells, progenitor cells, totipotent cells,pluripotent cells, induced pluripotent stem cells, undifferentiatedcells, differentiated cells, differentiating cells,trans-differentiating cells, cells from an adult, cells from a child,germ cells, circulating cells, resident cells, adherent cells, malignantcells, tumor cells, proliferating cells, quiescent cells, senescentcells, apoptotic cells, cytokine-producing cells, migrating cells, or acombination thereof In some embodiments, the bio-ink comprises aplurality of cells that express cell adhesion molecules. In someembodiments, cell adhesion molecules are selected from one or more of anadherin, a cadherin, a calsyntenin, a claudin, a cluster differentiationprotein, a contactin, an immunoglobulin, an integrin, a lectin, anectin, an occludin, a vinculin, a porimin, a podoplanin, a podocalyxin,a periostin, a neurotrimin, a neurexin, and a selectin. In someembodiments, the cell adhesion molecule is a receptor. In someembodiments, the cell adhesion molecule is a transmembrane protein.

In some embodiments, the plurality of cells comprises a geneticmutation. In some embodiments, the plurality of cells comprises anaturally-occurring genetic mutation. In some embodiments, thenaturally-occurring genetic mutation is a germline genetic mutation or asomatic genetic mutation. In some embodiments, the plurality of cellscomprises an induced genetic mutation. In some embodiments, the inducedgenetic mutation comprises a random genetic mutation or a targetedgenetic mutation. In some embodiments, one or more genes in theplurality of cells comprise a genetic mutation. In some embodiments, 2,3, 4, 5, 6, 7, 8, 9 or 10 genes in the plurality of cells comprise agenetic mutation. In some embodiments, more than 10 genes in theplurality of cells comprise a genetic mutation. In some embodiments, agene comprises a plurality of genetic mutations. In some embodiments,the plurality of cells has been genetically modified. In someembodiments, the plurality of cells is transfected with a nucleic acid.In some embodiments, the cells have been infected by a virus comprisinga nucleic acid. In some embodiments, the plurality of cells has beentransduced by a virus comprising a nucleic acid. In some embodiments,the virus is selected from a retrovirus, adenovirus or adeno-associatedvirus. In some embodiments, the nucleic acid is selected from a vector,a plasmid, a gene, a non-coding nucleic acid, an exon, an intron, adouble stranded DNA, a single stranded DNA, a RNA, a siRNA or a miRNA.In some embodiments, the nucleic acid is a gene. In some embodiments,the gene is a eukaryotic gene. In some embodiments, the gene is aprokaryotic gene. In some embodiments, the nucleic acid encodes a labelor an affinity tag.

In some embodiments, the plurality of cells comprises one or morelabels. In some embodiments, the one or more labels comprise afluorescent probe. In some embodiments, the fluorescent probe isselected from a CellTrace™ or CellTracker™ (Life Technologies, Carlsbad,CA, USA). In some embodiments, the label comprises a fluorescent tag. Insome embodiments, the fluorescent tag is mPlum, mCherry, tdTomato,mStrawberry, J-Red, DsRed-monomer, mOrange, mKO, mCitrine, Venus, YPet,EYFP, Emerald EGFP, CyPet, mCFPm, Cerulean, T-Sapphire, GFP or YFP. Insome embodiments the plurality of cells comprises an affinity tag. Insome embodiments, the affinity tag is a peptide. In some embodiments,the peptide is myc-tag, c-myc tag, FLAG-tag, His-tag, polyhistidine tag,HA-tag, V5, VSVG, softag 1, softag 3, express tag, S tag, fluoresceinisothiocyanate (FITC), dinitrophenyl, trinitrophenyl, peridininchlorophyll protein complex, biotin, phycoerythrin (PE), streptavidin,avidin, horse radish peroxidase (HRP), palmitoylation, nitrosylation,alkaline phosphatase, glucose oxidase, glutathione-S-transferase (GST),SUMO tag, thioredoxin, poly(NANP), poly-Arg, calmodulin binding protein,PurF fragment, ketosteroid isomerase, PaP3.30, TAF12 histone folddomain, maltose binding protein, or a fragment thereof. In someembodiments, the plurality of cells is from a tissue bank. In someembodiments, the plurality of cells is frozen or previously frozen. Insome embodiments, the plurality of cells are harvested or isolated froma donor tissue. In some embodiments, the donor tissue is harvested froma live animal. In some embodiments, the donor tissue is derived from amonkey, an ape, a gorilla, a chimpanzee, a cow, a horse, a dog, a cat, agoat, a sheep, a pig, a rabbit, a chicken, a turkey, a guinea pig, a rator a mouse. In some embodiments, the donor tissue is synthetic. In someembodiments, the plurality of cells is harvested from a live humandonor. In some embodiments, the plurality of cells is derived from theindividual. In some embodiments, the donor tissue is harvested from acadaver. In some embodiments, the plurality of cells is harvested from acadaver. In some embodiments, wherein the plurality of cells isharvested from a cadaver, the plurality of cells is harvested less thanabout 1 hour, less than about 2 hours, less than about 4 hours, lessthan about 6 hours, less than about 12 hours, less than about 24 hours,less than about 36 hours, less than about 48 hours, less than about 72hours after death. In some embodiments, the plurality of cells isharvested from a cadaver less than about 72 hours after death. In someembodiments, the plurality of cells is harvested from a cadaver between22 h and 72 h after death. In some embodiments, the plurality of cellsis treated with an antibiotic and/or an antimycotic after or while theyare isolated or harvested. In some embodiments, the antibiotic ispenicillin, streptomycin, actinomycin D, ampicillin, blasticidin,carbenicillin, cefotaxime, fosmidomycin, gentamicin, kanamycin,neomycin, polymyxin B, or any combination thereof. In some embodiments,the antimycotic is amphotericin B, nystatin, natamycin or anycombination thereof.

In some embodiments, the plurality of cells is propagated or maintainedin a cell culture media after they are isolated and before they arebioprinted. In some embodiments, cell culture media comprises essentialnutrients, growth factors, salts, minerals, vitamins, platelet-richplasma, or a combination thereof. In some embodiments, particularingredients are selected to enhance cell growth, differentiation orsecretion of specific proteins. In some embodiments, cell culture mediacomprises cellular differentiation agents. In some embodiments, theplurality of cells is cultured with a supernatant or conditioned mediafrom another population of cells in cell culture. In some embodiments,the plurality of cells are cultured in an atmosphere of about 1%, about2%, about 3%, about 5%, about 7%, about 10% or about 20% O₂. In someembodiments, cells are cultured in an atmosphere of about 1%, about 2%,about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9% orabout 10% CO₂. In some embodiments, cells are cultured at a temperatureof about 30° C., about 32° C., about 33° C., about 34° C., about 35° C.,about 36° C., about 37° C., about 38° C., about 39° C., about 40° C. orabout 42° C. In some embodiments, human chondrocytes are preferablycultured at approximately 37° C. with humidified air containing 5% CO₂,media changed about every four days. In some embodiments, the pluralityof cells are used for bioprinting when they grow to about 10%, about20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%,about 90%, about 100% confluence. In some embodiments the bio-ink ispreferably maintained at a temperature of about 37° C.In someembodiments, the plurality of cells comprises human chondrocytes. Insome embodiments, human chondrocytes are used for bioprinting when theygrow to about 80% to 90% confluence. In some embodiments, the pluralityof cells comprises chondrocytes, fibro-chondrocytes, or chondrogenicprecursors. In some embodiments, the bio-ink composition comprises aplurality of chondrogenic precursors. In some embodiments, the bio-inkcomposition comprises a plurality of fibro-chondrocytes.

In some embodiments, the chondrocytes are derived from human embryonicstem cells (hESCs) (i.e. hESC-derived chondrocytes). In someembodiments, the bio-ink composition comprises a plurality ofhESC-derived chondrocytes. In some embodiments, embryonic stem cellsinclude any commercially available or well established ESC cell linesuch as H9, H1, H7, or SA002. In some embodiments, the chondrocytes arederived from non-human embryonic stem cells. In some embodiments, thechondrocytes are derived from induced pluripotent stem cells (iPSCs)(i.e. iPSC-derived chondrocytes). In some embodiments, the bio-inkcomposition comprises a plurality of iPSC-derived chondrocytes. In someembodiments, the chondrocytes are derived from mesenchymal stem cells(MSCs). In some embodiments, the bio-ink composition comprises aplurality of MSC-derived chondrocytes. In some embodiments, thechondrocytes are derived from mesenchymal stem cells (MSCs) that are notadult bone marrow MSCs. In some embodiments, the bio-ink compositioncomprises a plurality of chondrocytes derived from MSCs that are notbone marrow derived-MSCs. In some embodiments, the bio-ink compositioncomprises a plurality of chondrocytes derived from MSCs that are notadult bone marrow derived-MSCs. In some embodiments, chondrocytes arederived from hESC-derived mesenchymal stem cells (MSCs). In someembodiments, the bio-ink composition comprises a plurality ofchondrocytes derived from hESC-derived MSCs. In some embodiments,chondrocytes are derived from H9 hESC-derived mesenchymal stem cells(MSCs). In some embodiments, the bio-ink composition comprises aplurality of chondrocytes derived from H9 hESC-derived MSCs. In someembodiments, chondrocytes are derived from H1 hESC-derived mesenchymalstem cells (MSCs). In some embodiments, the bio-ink compositioncomprises a plurality of chondrocytes derived from H1 hESC-derived MSCs.In some embodiments, chondrocytes are derived from H7 hESC-derivedmesenchymal stem cells (MSCs). In some embodiments, the bio-inkcomposition comprises a plurality of chondrocytes derived from H7hESC-derived MSCs. In some embodiments, chondrocytes are derived fromSA002 hESC-derived mesenchymal stem cells (MSCs). In some embodiments,the bio-ink composition comprises a plurality of chondrocytes derivedfrom SA002 hESC-derived MSCs. In some embodiments, chondrocytes arederived from iPSC-derived mesenchymal stem cells (MSCs). In someembodiments, the bio-ink composition comprises a plurality ofchondrocytes derived from iPSC-derived MSCs.

In some embodiments, the chondrocytes are derived from MSC-like cells.In some embodiments, the bio-ink composition comprises a plurality ofchondrocytes derived MSC-like cells. In some embodiments, MSC-like cellsare defined as cells that express one or more markers selected from:CD44, CD151, SOX5, SOX6, and SOX9. In some embodiments, the populationof MSC-like cells is at least 85% positive for CD73 and CD105. In someembodiments, the population of MSC-like cells is at least 95% positivefor CD73. In some embodiments, the population of MSC-like cells iscapable of multi-lineage differentiation into various tissues ofmesenchymal origin. In some embodiments, the population of MSC-likecells is capable of differentiating into cells of adipogenic,osteogenic, chondrogenic, and myogenic lineages. In some embodiments,the population of MSC-like cells is capable of differentiating intoadipocytes, chondrocytes, osteoblasts, and myocytes.

In some embodiments, the bio-ink composition comprises a plurality ofchondrocytes derived from a plurality of chondrogenic precursors. Insome embodiments, the bio-ink composition comprises a plurality ofchondrogenic precursors. In some embodiments, the bio-ink compositioncomprises a mixture comprising a plurality of chondrocytes and aplurality of chondrogenic precursors. In some embodiments, thechondrogenic precursors express one or more markers selected from:aggrecan, Collagen II, collagen type 2A1, Collagen IV, and SOX9. In someembodiments, chondrogenic precursors are derived from stem cells (SCs).In some embodiments, chondrogenic precursors differentiate intochondrocytes. In some embodiments, chondrogenic precursors are capableof differentiating into chondrocytes only. In some embodiments,chondrogenic precursors are not capable of differentiating into a celltype that is not a chondrocyte. In some embodiments, chondrogenicprecursors are not capable of differentiating into hypertrophicchondrocytes. In some embodiments, chondrogenic precursors do notdifferentiate into cells of adipogenic lineage, osteogenic lineage,myogenic lineages, or any combination thereof. In some embodiments,chondrogenic precursors do not differentiate into osteoblasts,osteocytes, osteoclasts, or any other type of bone cell. In someembodiments, chondrogenic precursors do not differentiate intoadipocytes, monovacuolar cells, plurivacuolar cells, or any other typeof fat cell. In some embodiments, chondrogenic precursors do notdifferentiate into myocytes, cardiomyocytes, skeletal myocytes, smoothmuscle cells, or any other type of muscle cell.

In some embodiments, chondrogenic precursors are derived frompluripotent stem cells. In some embodiments, chondrogenic precursors arederived from human embryonic stem cells (hESCs). In some embodiments,chondrogenic precursors are derived from induced pluripotent stem cells(iPSCs). In some embodiments, chondrogenic precursors are derived frommesenchymal stem cells (MSCs). In some embodiments, chondrogenicprecursors are derived from MSCs that are not adult bone marrowmesenchymal stem cells (MSCs). In some embodiments, chondrogenicprecursors are derived from hESC-derived mesenchymal stem cells (MSCs).In some embodiments, chondrogenic precursors are derived from H9hESC-derived mesenchymal stem cells (MSCs). In some embodiments,chondrogenic precursors are derived from H1 hESC-derived mesenchymalstem cells (MSCs). In some embodiments, chondrogenic precursors arederived from H7 hESC-derived mesenchymal stem cells (MSCs). In someembodiments, chondrogenic precursors are derived from SA002 hESC-derivedmesenchymal stem cells (MSCs). In some embodiments, chondrogenicprecursors are derived from iPSC-derived mesenchymal stem cells (MSCs).In some embodiments, the morphology of chondrogenic precursors isdescribed as rounded or elongated with relatively lower cytoplasm tonuclear ratio.

In some embodiments, the chondrocytes or chondrogenic precursors aremaintained in a cell culture comprising a growth factor. In someembodiments, the chondrocytes or chondrogenic precursors are maintainedin a cell culture media comprising transforming growth factor-β3(TGF-β3), basic fibroblast growth factor (bFGF), insulin-like growthfactor-1 (IGF-1), bone morphogenetic factors (BMPs), platelet derivedgrowth factors (PDGF), epidermal growth factor (EGF), or a combinationthereof. In some embodiments, the chondrocytes or chondrogenicprecursors are maintained in a cell culture media comprising BMP-1,BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8a, BMP-8b, BMP-10,BMP-15, or any combination thereof. In some embodiments, thechondrocytes or chondrogenic precursors are maintained in a cell culturemedia comprising PDGFA, PDGFB, PDGFC, PDGFD, PDGFRA, PDGFRB, or anycombination thereof. In some embodiments, the chondrocytes orchondrogenic precursors are cultured in a three dimensional cellculture. In some embodiments, the three dimensional culture comprises athree dimensional matrix. In some embodiments, the three dimensionalmatrix is a gel matrix. In some embodiments, the gel matrix isMatrigel®. In some embodiments, the three-dimensional matrix comprisescollagen, proteoglycan, fibrin, hyaluronic acid, poly-D-lactide,poly-L-lactide, poly-DL-lactide, polyglycolic acid, polylactic acid,hydroxyapatite, calcium phosphate, atelocollagen, fibrin, alginate, agarand/or gelatin. In some embodiments, the three-dimensional matrixcomprises collagen. In some embodiments, the collagen is cross-linked.In some embodiments, the collagen is solubilized. In some embodiments,the three-dimensional matrix comprises proteoglycan. In someembodiments, the three dimensional culture comprises pellet culture. Insome embodiments, the chondrocytes or chondrogenic precursors arecultured condensed together, for example, as a packed or pelleted cellmass under gentle centrifugation.

In some embodiments, the bio-ink comprises a plurality of stem cells. Insome embodiments, the bio-ink comprises a plurality of pluripotent stemcells. In some embodiments, the pluripotent stem cells are derived fromchondrocytes. In some embodiments, the pluripotent stem cells arederived from autologous chondrocytes. In some embodiments, thepluripotent stem cells are derived from allogeneic chondrocytes. In someembodiments, the bio-ink comprises a plurality of embryonic stem cells.In some embodiments, the bio-ink comprises a plurality of humanembryonic stem cells (hESCs). In some embodiments, the bio-ink comprisesa plurality of non-human embryonic stem cells. In some embodiments, thebio-ink comprises a plurality of induced pluripotent stem cells (iPSCs).In some embodiments, the bio-ink comprises a plurality of MSCs. In someembodiments, the bio-ink comprises a plurality of MSCs, wherein the MSCsare not adult bone marrow MSCs. In some embodiments, the bio-inkcomprises a plurality of MSCs, wherein the MSCs are derived frompluripotent stem cells. In some embodiments, the bio-ink comprises aplurality of MSCs, wherein the MSCs are derived from iPSCs. In someembodiments, the bio-ink comprises a plurality of MSCs, wherein the MSCsare derived from hESCs. In some embodiments, the bio-ink comprises aplurality of MSCs, wherein the MSCs are derived from H9 hESCs. In someembodiments, the bio-ink comprises a plurality of MSCs, wherein the MSCsare derived from H1 hESCs. In some embodiments, the bio-ink comprises aplurality of MSCs, wherein the MSCs are derived from H7 hESCs. In someembodiments, the bio-ink comprises a plurality of MSCs, wherein the MSCsare derived from SA002 hESCs. In some embodiments, the bio-ink comprisesa plurality of MSCs, wherein the MSCs are derived from H1 hESCs. In someembodiments, the bio-ink comprises a plurality of MSC-like cells, asdefined supra.

In some embodiments, the bio-ink comprises a cell culture medium. Insome embodiments, cell culture media is selected from Balanced Salts,Dulbecco’s Modified Eagle’s Medium, Dulbecco’s Modified Eagle’sMedium/Nutrient F-12 Media, Ham’s F-10 Media, Ham’s F-12 Media, MinimumEssential Medium Eagle, Medium 199, RPMI-1640 Medium, Ames’ Media, BGJbMedium (Fitton-Jackson Modification), Click’s Medium, CMRL-1066 Medium,Fischer’s Medium, Glascow Minimum Essential Medium (GMEM), Iscove’sModified Dulbecco’s Medium (IMDM), L-15 Medium (Leibovitz), McCoy’s 5AModified Medium, NCTC Medium, Swim’s S-77 Medium, Waymouth Medium,William’s Medium E, or combinations thereof. In some embodiments, thecell culture medium comprises a biological serum. In some embodiments,the serum is fetal bovine serum, fetal calf serum, fetal goat serum orhorse serum. In some embodiments, the biological serum content of thecell culture medium is about 0.5% v/v, about 1% v/v, about 2% v/v, about5% v/v, about 10% v/v, about 15% v/v, about 20% v/v, about 50% v/v,about 99% v/v, about 100% v/v. In some embodiments, the cell culturemedium comprises a buffering agent. In some embodiments the bufferingagent is selected from MES, ADA, PIPES, ACES, MOPSO, MOPS, BES, TES,HEPES, DIPSO, Acetamidoglycine, TAPSO, POPSO, HEPPSO, HEPPS, Tricine,Glycinamide, Bicine or TAPS.

In some embodiments, the bio-ink comprises a growth factor. In someembodiments, the growth factor is selected from Adrenomedullin (AM),Angiopoietin (Ang), Autocrine motility factor, Bone morphogeneticproteins (BMPs), Brain-derived neurotrophic factor (BDNF),Colony-stimulating factor (CSF), Epidermal growth factor (EGF),Erythropoietin (EPO), basic Fibroblast growth factor (bFGF), Glial cellline-derived neurotrophic factor (GDNF), Granulocyte colony-stimulatingfactor (G-CSF), Granulocyte macrophage colony-stimulating factor(GM-CSF), Growth differentiation factor-9 (GDF9), Hepatocyte growthfactor (HGF), Hepatoma-derived growth factor (HDGF), insulin,Insulin-like growth factor (IGF), Migration-stimulating factor,Myostatin (GDF-8), Nerve growth factor (NGF) and other neurotrophins,Platelet-derived growth factor (PDGF), Thrombopoietin (TPO),Transforming growth factor alpha(TGF-a), Transforming growth factorbeta(TGF-β), Tumor necrosis factor-alpha(TNF-a), Vascular endothelialgrowth factor (VEGF), placental growth factor (P1GF), Fetal BovineSomatotrophin (FBS), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7 or acombination thereof. In some embodiments, the bio-ink comprises TGF-β1and bFGF.

In some embodiments, the bio-ink comprises a component of extracellularmatrix. In some embodiments, the component of extracellular matrixcomprises a structural protein, a specialized protein, aglycosaminoglycan (GAG), a proteoglycan, or a combination thereof. Insome embodiments, a structural protein comprises collagen, elastin, andfibrillin. In some embodiments, the collagen comprises collagen type I,collagen type IL collagen type III, collagen type IV, collagen type V,collagen type VI, collagen type VII, collagen type VIII, collagen typeIX, collagen type X, collagen type XI, collagen type XII, collagen typeXIII, collagen type XIV, collagen type XV, collagen type XVI, collagentype XVII, collagen type XVIII, collagen type XIX, collagen type XX,collagen type XXI, collagen type XXII, collagen type XXIII, collagentype XXIV, collagen type XXV, collagen type XXVI, collagen type XXVII,collagen type XXVIII, collagen type XXIX or a combination thereof. Insome embodiments, the specialized protein comprises fibronectin,laminin, fibrinogen, tenascin, thrombospondin, integrin, or acombination thereof. In some embodiments, the glycosaminoglycancomprises a repeating disaccharide unit. In some embodiments, thedisaccharide unit comprises a modified sugar and hexuronic acid. In someembodiments, the modified sugar comprises N-acetylgalactosamine(GalNAc), N-acetylglucosamine (GlcNAc), or a combination thereof. Insome embodiments, the hexuronic acid comprises glucuronate (GlcA) oriduronate (IdA). In some embodiments, the glycosaminoglycan compriseshyaluronic acid, dermatan sulfate, chondroitin sulfate, heparin, heparinsulfate, and keratin sulfate. In some embodiments, the glycosaminoglycanis linked to core proteins, forming a proteoglycan. In some embodiments,the core proteins are rich in serine (Ser) and threonine (Thr) residues.In some embodiments, the proteoglycan comprises a tetrasaccharide linkercomprising a glucuronic acid (GlcA) residue, two galactose (Gal)residues, and a xylose (Xyl) residue. In some embodiments, theextracellular matrix is derived from a human, a cow, a horse, a sheep, agoat, a chimpanzee, a monkey, a rat, a pig, a mouse, a rabbit, or asynthetic reaction.

In some embodiments, the bio-ink comprises a synthetic polymer, anatural polymer, or a combination thereof. In some embodiments, thebio-ink is a gel. In some embodiments, the gel is a biogel or ahydrogel. In some embodiments, the synthetic polymer is polylactide(PLA), polycaprolactone (PCL), polyethylene glycol (PEG), a PEGmacromer, polyethylene glycol methacrylate (PEGMA), polyethylenedimethacrylate (PEGDMA), poly(hydroxyethyl methacrylate) (PHEMA),polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), carboxymethylcellulose (CMC), polyimide (PI), polyacrylate (PAA), polyurethane (PU),PEG-lactide, PEG-glycolide or a combination thereof. In someembodiments, the gel comprises a PEGDMA hydrogel. In some embodiments,the PEGDMA polymer is 10% w/v hydrogel. In some embodiments, the PEGDMApolymer is 20% w/v hydrogel. In some embodiments, the gel does notcomprise a synthetic polymer. In some embodiments, PEG macromerscomprise reactive chain ends such as acrylate, methacrylate, allylether, maleimide, vinyl sulfone, N-hydroxysuccinimide (NHS) ester andvinyl ether groups. In some embodiments, the alcohol chain ends of PEGare esterified using acid chlorides (e.g., acryloyl chloride,methacryloyl chloride) in the presence of base. In some embodiments, PEGchain ends are etherified under basic conditions by reaction with alkylhalides such as 2-chloroethyl vinyl ether or allyl bromide. In someembodiments, acrylate, methacrylate, vinyl sulfone, maleimide, vinylether and allyl ether are capable of step growth network formation orpolymerization. In some embodiments, polymerization of macromers isinitiated using redox-generated radicals (e.g., ammonium persulfate andTEMED), or radicals generated with light. In some embodiments, thenatural polymer is alginate, cellulose, gelatin, pectin, chitosan,paraffin, agarose, or a combination thereof. In some embodiments, thebio-ink comprises Matrigel®.

In some embodiments, the synthetic polymer or the natural polymercomprises a modification to enable crosslinking. In some embodiments,the modification to enable crosslinking is methacrylation. In someembodiments, the bio-ink comprises methacrylated collagen. In someembodiments, the bio-ink comprises methacrylated hyaluronic acid. Insome embodiments, the bio-ink comprises a methacrylated extracellularmatrix protein. In some embodiments, the synthetic polymer or thenatural polymer comprises a functional molecule. In some embodiments,the functional molecule comprises a bioactive protein or drug. In someembodiments, the synthetic polymer or the natural polymer comprises apeptide to promote cell adhesion, a peptide to promote proliferation, ora peptide to promote differentiation. In some embodiments, the peptideto promote cell adhesion is arginyl-glycyl-aspartic acid (RGD). In someembodiments, the synthetic polymer or the natural polymer comprises abiodegradable link. In some embodiments, the biodegradable link is amatrix metalloproteinase (MMP)-sensitive link or anaggrecanase-sensitive link.

In some embodiments, the bio-ink comprises an additional agent. In someembodiments, the additional agent comprises a salicylic acid, acarboxylic acid, a lipid or fatty acid, a surfactant, a starch, aparaffin, a silica, a glycerol, or a combination thereof. In someembodiments, the lipid or fatty acid comprises palmitic acid, oleicacid, linolenic acid, omega-3 fatty acid or a combination thereof.

In some embodiments, the bio-ink comprises a biochemical factor. In someembodiments, the biochemical factor is selected from an anticoagulant,albumin, selenium, an amino acid, a vitamin, a hormone, a mineral, orany combination thereof. In some embodiments, the bio-ink comprises aprotein. In some embodiments, the protein is a kinase, a hormone, acytokine, a chemokine, an anti-inflammatory factor, a pro-inflammatoryfactor, an apoptotic factor or a steroid. In some embodiments, thebio-ink comprises an enzyme. In some embodiments, the enzyme is aprotease, a collagenase, a nuclease, or a combination thereof. In someembodiments, the protease is a serine protease, a threonine protease, acysteine protease, an aspartate protease, a glutamic acid protease, ametalloprotease, an exopeptidase, an endopeptidase, a trypsin, achymotrypsin, a pepsin, a papain, an elastase, a carboxypeptidase, anaminopeptidase, a thrombase, a plasmin, a cathepsin, or snake venom.

In some embodiments, the bio-ink comprises a therapeutic agent. In someembodiments, the therapeutic agent is selected from an antibiotic and/oran antimycotic. In some embodiments, the antibiotic is penicillin,streptomycin, actinomycin D, ampicillin, blasticidin, carbenicillin,cefotaxime, fosmidomycin, gentamicin, kanamycin, neomycin, polymyxin B,or a combination thereof. In some embodiments, the antimycotic isamphotericin B, nystatin, natamycin or a combination thereof. In someembodiments, the therapeutic agent is selected from an anti-inflammatorytherapeutic agent. In some embodiments, the anti-inflammatorytherapeutic agent is a non-steroidal anti-inflammatory therapeuticagent. In some embodiments, the non-steroidal anti-inflammatorytherapeutic agent is a cyclooxygenase (COX) inhibitor. In someembodiments, the COX inhibitor is selected from a COX1 inhibitor, COX2inhibitor or combination thereof. In some embodiments, theanti-inflammatory therapeutic agent comprises a steroid. In someembodiments, the steroid is a glucocorticoid. In some embodiments, theglucocorticoid is dexamethasone.

In some embodiments, the method comprises positioning a light sourcewithin proximity of the internal tissue defect. In some embodiments, thelight source is a laser. In some embodiments, the light source is alamp. In some embodiments, the light source emits light in a focusedregion. In some embodiments, the light source emits light in a pattern.In some embodiments, the pattern of light cross-links the bio-ink. Insome embodiments, the method comprises a wash step to remove the bio-inkwhich was not cross-linked. In some embodiments, the light source isconnected to the endoscope. In some embodiments, the light source emitslight with a visible wavelength of to 400 nm to 700 nm. In someembodiments, the light source emits UV light. In some embodiments, UVlight comprises UV-A light, UV-B light, or UV-C light. In someembodiments, UV-A light comprises a wavelength of light between 315 nmand 400 nm. In some embodiments, UV-B light comprises a wavelength oflight between 280 nm and 315 nm. In some embodiment, UV-C lightcomprises a wavelength of light between 100 nm to 280 nm. In someembodiments, the light source is an LED.

In some embodiments, the bio-ink is photopolymerizable. In someembodiments, the bio-ink is photodegradable. In some embodiments, thebio-ink comprises photo-releasable factors. In some embodiments,photo-releasable factors are selected from cells, growth factors,proteases, ligands, hormones, extracellular matrix, cytokines,anti-inflammatory factors, pro-inflammatory factors, adhesion molecules,or a combination thereof. In some embodiments, photo-releasable factorsare used to form a feature of the bioprinted tissue (e.g. vasculature).In some embodiments, the bio-ink comprises a PEG with a degradable esterlinkage. In some embodiments, the bio-ink comprises a factor that isattached to a component of the bio-ink or the extracellular matrix. Insome embodiments, the factor is released by hydrolysis or enzymolysis ofa bond that attaches the factor to the component of the gel orextracelluar matrix. In some embodiments, the factor is released byhydrolysis or enzymolysis of the gel component or the extracellularmatrix. In some embodiments, the factor is released from the gelcomponent or the extracellular matrix by the enzyme. In someembodiments, the enzyme is present in the internal tissue defect. Insome embodiments, the factor released is a therapeutic agent or a growthfactor. In some embodiments, the growth factor induces angiogenesis uponrelease.

Bio-Ink Constructs

Provided herein are bio-ink constructs, wherein the bio-ink constructsare produced by the methods and systems disclosed herein.

In some embodiments, the bio-ink construct comprises a plurality ofbio-ink layers. In some embodiments, the bio-ink construct is a livingtissue.

In some embodiments, the bio-ink construct has elevatedglycosaminoglycan relative to respective cells in two dimensional cellculture and/or elevated proteoglycans relative to cells in twodimensional cell culture. In some embodiments, the average cellviability of the bio-ink construct is at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 98% or at least 99%. In some embodiments, theaverage cell viability of the bio-ink construct is about 90%. In someembodiments, the average cell viability of the bio-ink construct isabout 100%.

In some embodiments, the bio-ink construct comprises a plurality ofbio-ink layers. In some embodiments, each bio-ink layer comprises one ormore cells. In some embodiments, a bio-ink layer comprises two or morecells. In some embodiments, a plurality of bio-ink layers is printed onto or in an internal tissue defect. In some embodiments, the internaltissue defect is in a human. In some embodiments, the internal tissuedefect is in an animal. In some embodiments, the plurality of bio-inklayers is adjacent. In some embodiments, one or more cells of eachbio-ink layer are adjacent to one or more cells of an adjacent bio-inklayer. In some embodiments, the bio-ink layers are the same dimension.In some embodiments, the bio-ink layers are each independently asuitable dimension. In some embodiments, the bio-ink construct comprises2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100 or more bio-ink layers.In some embodiments, the thickness of each layer of the plurality ofbio-ink layers is independently about 10 µm, about 12 µm, about 14 µm,about 16 µm, about 18 µm, about 20 µm, about 30 µm, about 40 µm, about50 µm, about 60 µm, about 70 µm, about 80 µm, about 90 µm, about 100 µm,about 125 µm, about 150 µm, about 175 µm, about 200 µm, about 225 µm,about 250 µm, about 275 µm, about 300 µm, about 325 µm, about 350 µm,about 375 µm, about 400 µm, about 425 µm, about 450 µm, about 500 µm,about 525 µm, about 550 µm, about 575 µm, about 600 µm, about 625 µm,about 650 µm, about 675 µm, about 700 µm, about 725 µm, about 750 µm,about 775 µm, about 800 µm, about 825 µm, about 850 µm, about 875 µm,about 900 µm, about 925 µm, about 950 µm, about 975 µm, or about 1 mm.In some embodiments the thickness of each layer of the plurality ofbio-ink layers or live tissue of a construct is independently less thanabout 10 µm, less than about 12 µm, less than about 14 µm, less thanabout 16 µm, less than about 18 µm, less than about 20 µm, less thanabout 22 µm, less than about 24 µm, less than about 26 µm, less thanabout 28 µm or less than about 30 µm at its thinnest point.

In some embodiments, the bio-ink construct will be vascularized bysurrounding tissue. In some embodiments, the bio-ink construct isavascular. In some embodiments, the bio-ink construct will not bevascularized by surrounding tissue. In some embodiments, the bio-inkconstruct comprises cartilage. In some embodiments, the bio-inkconstruct consists essentially of cartilage. In some embodiments, thewater content of the cartilage is greater than about 20%, about 30%,about 40%, about 50%, about 60%, about 70%, about 72%, about 74%, about75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%,about 82%, about 83%, about 84%, about 85%, about 87%, or about 90%. Insome embodiments, the water content of the cartilage is about 80%.

Bioprinting Systems

Provided herein are bioprinting systems for producing bio-ink constructsdisclosed herein. Furthermore, these bioprinting systems may be used toperform methods of bioprinting disclosed herein. Bioprinting systems, asdescribed throughout this instant application, may be used with variousbio-ink compositions disclosed herein. In some embodiments, the systemsdescribed herein are biological composition delivery system. In someembodiments, the systems comprise a bioprinter, a control system, adisplay screen, a robotic arm, a light source, a sensor, an endoscope, athree dimensional scanner, or a combination thereof.

In some embodiments, the systems disclosed herein improve the accuracyand precision of placement and orientation of a surgical implant,bio-ink deposition, and bio-ink construct formation during surgery. Insome embodiments, the system only prints in the desired area ensuringsafer surgery. In some embodiments, the surgical implant is a bio-inkconstruct. In some embodiments, the surgical implant is a meniscalimplant produced by the methods described herein. In some embodiments,the systems disclosed herein enable for adjustments during surgeryinvolving tissue resection, deposition of a bio-ink, or positioning of abio-ink construct. In some embodiments, the systems disclosed herein,when used during a surgery, improve recovery time of the patient afterthe surgery. In some embodiments, the systems disclosed herein, whenused during a surgery, lower the amount of pain that the patient enduresafter the surgery.

Bioprinters

In some embodiments, the systems disclosed herein comprise a bioprinter.In some embodiments, the bioprinter comprises a printhead. In someembodiments, the printhead comprises a needle, an extended cylinder, afluid line, a print nozzle (FIG. 5A), or a plurality of print nozzles(FIG. 5B). In some embodiments, the bioprinter comprises a secondprinthead. In some embodiments, the printhead is attached to a roboticarm. In some embodiments, a plurality of printheads is attached to aplurality of robotic arms. In some embodiments, the robotic arm guidesthe printhead. In some embodiments, the robotic arm guides thebioprinter. In some embodiments, the robotic arm positions the printheadwithin proximity of or in contact with the internal tissue defect. Insome embodiments, the robotic arm positions the printhead withinproximity of or in contact with the chondral defect. In someembodiments, the robotic arm positions the printhead within proximity ofor in contact with curved or complex surfaces. In some embodiments, therobotic arm positions the bioprinter within proximity of or in contactwith the internal tissue defect. In some embodiments, the robotic armpositions the bioprinter within proximity of or in contact with thechondral defect. In some embodiments, the robotic arm positions thebioprinter within proximity of or in contact with curved or complexsurfaces.

In some embodiments, the size of the bioprinter enables the bioprinterto be inserted into the patient through an incision in the skin of thepatient. In some embodiments, the size of the printhead enables theprinthead to be inserted into the patient through an incision in theskin of the patient. In some embodiments, the diameter of the nozzleenables the nozzle to be inserted into the patient through an incisionin the skin of the patient. In some embodiments, the diameters of aplurality of nozzles enable the plurality of nozzles to be inserted intothe patient through an incision in the skin of the patient. In someembodiments, the diameter of the needle enables the needle to beinserted into the patient through an incision in the skin of thepatient.

In some embodiments, the size of the bioprinter enables the bioprinterto be inserted into the patient through an incision in the skin of thepatient during minimally invasive surgery, wherein the length of theincision is between about 1 mm and about 5 mm. In some embodiments, thesize of the printhead enables the printhead to be inserted into thepatient through an incision in the skin of the patient during minimallyinvasive surgery, wherein the length of the incision is between about 1mm and about 5 mm. In some embodiments, the diameter of the needleenables the needle to be inserted into the patient through an incisionin the skin of the patient during minimally invasive surgery, whereinthe length of the incision is between about 1 mm and about 5 mm. In someembodiments, the diameter of the nozzle enables the nozzle to beinserted into the patient through an incision in the skin of the patientduring minimally invasive surgery, wherein the length of the incision isbetween about 1 mm and about 5 mm. In some embodiments, the diameters ofthe plurality of nozzles enable the plurality of nozzles to be insertedinto the patient through an incision in the skin of the patient duringminimally invasive surgery, wherein the length of the incision isbetween about 1 mm and about 5 mm.

In some embodiments, the size of the bioprinter enables the bioprinterto be inserted into the patient through an incision in the skin of thepatient. In some embodiments, the length of the incision in the skin ofthe patient ranges from about 1 mm to about 10 mm. In some embodiments,the length of the incision in the skin of the patient ranges from about1 mm to about 5 mm. In some embodiments, the length of the incision inthe skin of the patient ranges from about 5 mm to about 10 mm. In someembodiments, the length of the incision in the skin of the patient isabout 1 mm. In some embodiments, the length of the incision in the skinof the patient is about 2 mm. In some embodiments, the length of theincision in the skin of the patient is about 3 mm. In some embodiments,the length of the incision in the skin of the patient is about 4 mm. Insome embodiments, the length of the incision in the skin of the patientis about 5 mm. In some embodiments, the length of the incision in theskin of the patient is about 6 mm. In some embodiments, the length ofthe incision in the skin of the patient is about 7 mm. In someembodiments, the length of the incision in the skin of the patient isabout 8 mm. In some embodiments, the length of the incision in the skinof the patient is about 9 mm. In some embodiments, the length of theincision in the skin of the patient is about 10 mm. In some embodiments,the length of the incision in the skin of the patient is between about10 mm to about 50 mm. In some embodiments, the length of the incision inthe skin of the patient is about 10 mm. In some embodiments, the lengthof the incision in the skin of the patient is about 20 mm. In someembodiments, the length of the incision in the skin of the patient isabout 30 mm. In some embodiments, the length of the incision in the skinof the patient is about 40 mm. In some embodiments, the length of theincision in the skin of the patient is about 50 mm.

In some embodiments, the printhead comprises a plurality of printnozzles. In some embodiments, the plurality of print nozzles is arrangedin an array. In some embodiments, the plurality of print nozzles isindependently controlled and actuated. In some embodiments, theplurality of print nozzles is actuated to eject an individual droplet ofthe bio-ink. In some embodiments, the plurality of print nozzles ejectsthe individual droplet of bio-ink simultaneously. In some embodiments,the plurality of print nozzles ejects the individual droplet in aspecified sequence. In some embodiments, the plurality of nozzles isarranged in a variety of configurations that have the ability to conformto curved and complex surfaces (FIGS. 1A-1F).

In some embodiments, the printhead stores the bio-ink. In someembodiments, the printhead stores 100 µl, 200 µl, 300 µl, 400 µl, 500µl, 600 µl, 70 µl, 800 µl, 900 µl, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7ml, 8 ml, 9 ml, 10 ml, 20 ml, 30 ml, 40 ml, 50 ml, 60 ml, 70 ml, 80 ml,90 ml, 100 ml, 200 ml, 300 ml, 400 ml, or 500 ml of bio-ink. In someembodiments the printhead is disposable.

In some embodiments, the printhead comprises at least 5 print nozzles.In some embodiments, the printhead comprises about 6-20 print nozzles.In some embodiments, the printhead comprises 10, 12 or 16 print nozzles.In some embodiments, the printhead comprises about 5, about 10, about15, about 20, about 25, about 30, about 35, about 40, about 45, about50, about 55, about 60, about 65, about 70, about 75, about 80, about85, about 90, about 95 or about 100 print nozzles. In some embodimentsthe diameter of the print nozzle is about 5 µm, about 10 µm, about 15µm, about 20 µm, about 25 µm, about 30 µm, about 35 µm, about 40 µm,about 45 µm, about 50 µm, about 55 µm, about 60 µm, about 65 µm, about70 µm, about 75 µm, about 80 µm, about 85 µm, about 90 µm, about 95 µm,about 100 µm, about 105 µm, about 110 µm, about 115 µm, about 120 µm,about 125 µm, about 130 µm, about 135 µm, about 140 µm, about 145 µm, orabout 150 µm. In some embodiments, the diameter of the print nozzle isabout 120 µm. In some embodiments, the diameter of the print nozzle isless than about 120 µm. In some embodiments, the diameter of the printnozzle is greater than 120 µm. In some embodiments, the printhead is amodified inkjet printhead. In some embodiments, the inkjet printhead isa thermal inkjet printhead. In some embodiments, the inkjet printhead isa piezoelectric inkjet printhead. In some embodiments, the printhead isa modified custom printhead. In some embodiments, the printhead is acustom printhead produced for bioprinting. In some embodiments, theprinthead is a modified laser printhead.

In some embodiments, the print nozzles are arranged in one row of printnozzles. In some embodiments, the print nozzles are arranged in two rowsof print nozzles. In some embodiments, the print nozzles are arranged inabout 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 rows ofprint nozzles. In some embodiments, the print nozzles are arranged intwo rows of 10, 12 or 16 print nozzles. In some embodiments, each row ofprint nozzles has the same number of print nozzles. In some embodiments,one or more rows of print nozzles have a different number of printnozzles from one or more other rows. In some embodiments, the length ofthe rows is described as the Y-axis of the row. In some embodiments, theY-axis spacing between print nozzles in a row of print nozzles isbetween about 5 micrometers and about 500 micrometers. In someembodiments, the Y-axis spacing between print nozzles in a row of printnozzles is between about 5 micrometers and about 200 micrometers,between about 5 micrometers and about 100 micrometers, between about 50micrometers and about 200 micrometers, between about 1 micrometer andabout 50 micrometers, or between about 200 micrometers and about 400micrometers. In some embodiments, the Y-axis spacing between printnozzles in a row of print nozzles is between about 200 micrometers andabout 400 micrometers. In some embodiments, the spacing between rows ofprint nozzles is between about 5 micrometers and about 500 micrometers.In some embodiments, the spacing between rows of print nozzles isbetween about 5 micrometers and about 200 micrometers, between about 5micrometers and about 100 micrometers, between about 50 micrometers andabout 200 micrometers, between about 1 micrometer and about 50micrometers, or between about 200 micrometers and about 400 micrometers.In some embodiments, the spacing between rows of print nozzles isbetween about 200 micrometers and about 400 micrometers. In someembodiments of 10 print nozzles arranged in two rows, the Y-axis spacingbetween print nozzles is about 20 dpi, about 30 dpi, about 40 dpi, about60 dpi, about 70 dpi, about 80 dpi, about 90 dpi, about 100 dpi, about110 dpi, about 120 dpi, about 130 dpi, about 140 dpi, about 150 dpi,about 160 dpi, about 170 dpi, about 180 dpi, about 190 dpi, about 200dpi, about 250 dpi or about 300 dpi. In some embodiments of 10 printnozzles arranged in two rows, the Y-axis spacing between print nozzlesis about 150 dpi. In some embodiments, the spacing between rows is about200 gm, about 220 gm, about 240 gm, about 260 gm, about 280 gm, about300 gm, about 320 gm, about 340 gm, about 360 gm, about 380 gm, about400 gm, about 420 gm, about 450 gm, or about 500 gm. In someembodiments, the spacing between rows is about 340 gm. In someembodiments of 12 print nozzles arranged in two rows, the Y-axis spacingbetween print nozzles is about 20 dpi, about 30 dpi, about 40 dpi, about60 dpi, about 70 dpi, about 80 dpi, about 90 dpi, about 100 dpi, about110 dpi, about 120 dpi, about 130 dpi, about 140 dpi, about 150 dpi,about 160 dpi, about 170 dpi, about 180 dpi, about 190 dpi, about 200dpi, about 250 dpi or about 300 dpi. In some embodiments of 12 printnozzles arranged in two rows, the Y-axis spacing between print nozzlesis about 180 dpi or 141 µm. In some embodiments, the spacing betweenrows is about 200 µm, about 220 µm, about 240 µm, about 260 µm, about280 µm, about 300 µm, about 320 µm, about 340 µm, about 360 µm, about380 µm, about 400 µm, about 420 µm, about 450 µm, or about 500 pm. Insome embodiments, the spacing between rows is about 300 µm. In someembodiments of 16 print nozzles arranged in two rows, the Y-axis spacingbetween print nozzles is about 20 dpi, about 30 dpi, about 40 dpi, about60 dpi, about 70 dpi, about 80 dpi, about 90 dpi, about 100 dpi, about110 dpi, about 120 dpi, about 130 dpi, about 140 dpi, about 150 dpi,about 160 dpi, about 170 dpi, about 180 dpi, about 190 dpi, about 200dpi, about 250 dpi, about 300 dpi, about 320 dpi, about 340 dpi, about360 dpi, about 380 dpi, about 400 dpi, about 450 dpi or about 500 dpi.In some embodiments of 16 print nozzles arranged in two rows, the Y-axisspacing between print nozzles is about 300 dpi or 84 µm. In someembodiments, the spacing between rows is about 200 µm, about 220 µm,about 240 µm, about 260 µm, about 280 µm, about 300 µm, about 320 µm,about 340 µm, about 360 µm, about 380 µm, about 400 µm, about 420 µm,about 450 µm, or about 500 µm. In some embodiments, the spacing betweenrows is about 230 µm.

In some embodiments, the printhead comprises a configuration of printnozzles. In some embodiments, the configuration of print nozzlescomprises parallel print nozzles. In some embodiments, the configurationcomprises non-parallel print nozzles. In some embodiments, theconfiguration comprises converging print nozzles (FIGS. 1A and 1D). Insome embodiments, the configuration comprises diverging print nozzles(FIGS. 1B and 1E). In some embodiments, the print nozzles are positionedat a level above or at the internal tissue defect. In some embodiments,the level is changed so as to increase or decrease proximity of theprint nozzle to the internal tissue defect (FIGS. 1C and 1F). In someembodiments, the configuration comprises print nozzles with the level ofthe print nozzles all in the same plane (FIGS. 1C and 1F). In someembodiments, the configuration comprises print nozzles with the level ofone or more print nozzles not in the same plane. In some embodiments,one or more configurations are combined. In some embodiments, theplurality of nozzles is arranged in a specific shape (FIGS. 2A-2E).

In some embodiments, the configuration of print nozzles comprises aprint nozzle, wherein the direction of the print nozzle is modular. Insome embodiments, the print nozzle permits drop volumes of bio-inkejected from a printhead between about 2 picoliters and about 220picoliters. In some embodiments, a bio-ink drop is about 1 pL, about 2pL, about 5 pL, 10 pL, about 15 pL, about 20 pL, about 25 pL, about 30pL, about 35 pL, about 40 pL, about 45 pL, about 50 pL, about 55 pL,about 60 pL about 65 pL, about 70 pL, about 75 pL, about 80 pL, about 85pL, about 90 pL, about 95 pL, about 100 pL, about 105 pL, about 110 pL,about 115 pL, about 120 pL, about 125 pL, about 130 pL, about 135 pL,about 140 pL, about 145 pL, about 150 pL, about 155 pL, about 160 pL,about 165 pL, about 170 pL, about 175 pL, about 180 pL, about 185 pL,about 190 pL, about 195 pL, about 200 pL, about 250 pL, about 300 pL,about 500 pL, or about 1 nL. In some embodiments, the printhead has aresolution of at least about 100 dots per inch (dpi). In someembodiments, the printhead has a resolution of at least about 150 dpi,at least about 200 dpi, at least about 300 dpi, at least about 400 dpi,at least about 500 or more dpi, or about 1000 or more dpi. In someembodiments, the print nozzles fire with a frequency of about 1000 Hz,about 1200 Hz, about 1400 Hz, about 1600 Hz, about 1800 Hz, about 2000Hz, about 2200 Hz, about 2400 Hz, about 2600 Hz, about 2800 Hz, about3000 Hz, about 3200 Hz, about 3400 Hz, about 3600 Hz, about 3800 Hz,about 4000 Hz, about 4200 Hz, about 4400 Hz, about 4600 Hz, about 5000Hz, about 5200 Hz, about 5400 Hz, about 5600 Hz, about 5800 Hz, or about6000 Hz.

In some embodiments, the print nozzle is a coaxial nozzle. In someembodiments, the coaxial nozzle comprises two concentric nozzlescomprising a first nozzle and a second nozzle. In some embodiments, thefirst nozzle is the inner nozzle and the second nozzle is the outernozzle. In some embodiments, the first nozzle and the second nozzleeject a first bio-ink and a second bio-ink, respectively. In someembodiments, the first nozzle and the second nozzle eject bio-ink usingdifferent bio-ink printing parameters. In some embodiments, the coaxialnozzle comprises three concentric nozzles comprising a first nozzle, asecond nozzle, and a third nozzle. In some embodiments, the first nozzleis the inner nozzle, the second nozzle is the middle nozzle, and thethird nozzle is the outer nozzle. In some embodiments, the first nozzle,second nozzle, and third nozzle eject a first bio-ink, a second bio-ink,and a third bio-ink, respectively. In some embodiments, the firstbio-ink, the second bio-ink, or the third bio-ink comprises thecross-linking agent. In some embodiments, the first bio-ink, the secondbio-ink, or the third bio-ink comprises the synthetic polymer or thenatural polymer. In some embodiments, the first bio-ink, the secondbio-ink, or the third bio-ink comprises the plurality of cells. In someembodiments, the first nozzle, the second nozzle, and the third nozzleeject bio-ink using different bio-ink printing parameters. In someembodiments, the coaxial nozzle comprises four or more concentricnozzles. In some embodiments, the coaxial nozzle is used to create avasculature in the bio-ink construct.

In some embodiments, the bioprinter comprises a plurality of printheads.In some embodiments, the plurality of printheads comprises a secondprinthead, a third printhead, a fourth printhead, a fifth printhead, asixth printhead, a seventh printhead, an eight printhead, a ninthprinthead, or a tenth printhead. In some embodiments, the plurality ofprint nozzles are of different sizes and diameters and are distributedin various locations (FIG. 2E). In some embodiments the plurality ofprint nozzles comprises coaxial nozzles, non-coaxial nozzles, or acombination thereof.

Control Systems

In some embodiments, the systems disclosed herein comprise a controlsystem. In some embodiments, the control system is connected to thebioprinter. In some embodiments, the control system is in communicationwith the bioprinter. In some embodiments, the control system haswireless communication with the bioprinter. In some embodiments, thecontrol system controls the bio-ink printing parameters of thebioprinter.

In some embodiments, the control system comprises a computer system. Insome embodiments, the control system comprises a robotic arm operativelyconnected to the computer system. In some embodiments, the controlsystem comprises a plurality of robotic arms operatively connected tothe computer system. In some embodiments, the plurality of robotic armscomprises a second robotic arm, a third robotic arm, a fourth roboticarm, a fifth robotic arm, a sixth robotic arm, a seventh robotic arm, aneight robotic arm, a ninth robotic arm, or a tenth robotic arm. In someembodiments, the robotic arm is able to move in any direction and in anyangle. In some embodiments, the robotic arm has six degrees of freedom.In some embodiments, the robotic arm has five degrees of freedom. Insome embodiments, the robotic arm has four degrees of freedom. In someembodiments, the robotic arm has three degrees of freedom. In someembodiments, the robotic arm has two degrees of freedom. In someembodiments, the robotic arm enables the bioprinter to be positioned atany angle with respect to the substrate being printed on. In someembodiments, the increased flexibility in movement of the robotic armenables the bioprinter to print directly onto a patient.

In some embodiments, the computer system comprises a computing device, amicrocontroller, a processor, a memory device, an operating system, anda software module for monitoring or operating the printhead. In someembodiments, the computer system comprises a computing device. In someembodiments, the computing device is a microcontroller. In someembodiments, the microcontroller is an 8-bit, 16-bit, or 32-bitmicrocontroller. In some embodiments, the microcontroller is an 8051microcontroller, a programmable interface controller (PIC), an AVR orAdvanced Virtual RISC microcontroller, or an ARM® microcontroller.

In some embodiments, the computing device is a desktop computer or alaptop computer. In some embodiments, the computing device is a mobiledevice. In some embodiments, the mobile device is a smart phone or asmart watch. In some embodiments, the computing device is a portabledevice. In accordance with the description herein, suitable computingdevices further include, by way of non-limiting examples, notebookcomputers, tablet computers, netbook computers, smart book computers,subnotebook computers, ultra-mobile PCs, handheld computers, personaldigital assistants, Internet appliances, smart phones, music players,and portable video game systems. Many mobile smart phones are suitablefor use in the systems described herein. Suitable tablet computersinclude those with booklet, slate, and convertible configurations.Suitable portable video game systems include, by way of non-limitingexamples, Nintendo DS™ and Sony® PSP™.

In some embodiments, the computer system comprises a computer programincluding instructions executable by the processor causing the processorto: 1) control the movement and position of the robotic arm, 2) controla bio-ink printing parameter, and 3) control the bio-printing process.In some embodiments, the computer system controls the bio-printingprocess by directing the bioprinter to the location on the substratewhere the bio-ink gets deposited.

In some embodiments, the computer system comprises a processor, a memorydevice, an operating system, and a software module for monitoring oroperating the printhead. In some embodiments, the computer systemcomprises a digital processing device and includes one or more hardwarecentral processing units (CPU). In further embodiments, the computersystem includes an operating system configured to perform executableinstructions. In some embodiments, the operating system is software,including programs and data, which manages the device’s hardware andprovides services for execution of applications. Those of skill in theart will recognize that suitable server operating systems include, byway of non-limiting examples, FreeBSD, OpenBSD, NetBSD®, Linux, Apple®Mac OS X Server®, Oracle® Solaris®, Windows Server®, and Novell®NetWare®. Those of skill in the art will recognize that suitablepersonal computer operating systems include, by way of non-limitingexamples, Microsoft® Windows®, Apple® Mac OS X®, UNIX®, and UNIX-likeoperating systems such as GNU/Linux®. In some embodiments, the operatingsystem is provided by cloud computing. Those of skill in the art willalso recognize that suitable mobile smart phone operating systemsinclude, by way of non-limiting examples, Nokia® Symbian® OS, Apple®iOS®, Research In Motion® BlackBerry OS®, Google°Android®, Microsoft®Windows Phone® OS, Microsoft® Windows Mobile® OS, Linux, and Palm® WebOS. In some embodiments, the computer system includes a storage and/ormemory device. In some embodiments, the storage and/or memory device isone or more physical apparatuses used to store data or programs on atemporary or permanent basis. In some embodiments, the device isvolatile memory and requires power to maintain stored information. Insome embodiments, the device is non-volatile memory and retains storedinformation when the digital processing device is not powered. Infurther embodiments, the non-volatile memory comprises flash memory. Insome embodiments, the non-volatile memory comprises dynamicrandom-access memory (DRAM). In some embodiments, the non-volatilememory comprises ferroelectric random access memory (FRAM). In someembodiments, the non-volatile memory comprises phase-change randomaccess memory (PRAM). In some embodiments, the device is a storagedevice including, by way of non-limiting examples, CD-ROMs, DVDs, flashmemory devices, magnetic disk drives, magnetic tapes drives, opticaldisk drives, and cloud computing based storage. In some embodiments, thestorage and/or memory device is a combination of devices such as thosedisclosed herein.

In some embodiments, the computer systems described herein include userinterfaces. In some embodiments, the input device is a keyboard. Infurther embodiments, the input device is a key pad. In a particularembodiment, the input device is a simplified key pad for use by asubject with communications limitations (e.g., due to age, infirmity,disability, etc.), wherein each key is associated with a color, a shape,and health/communication concept. In some embodiments, the input deviceis a pointing device including, by way of non-limiting examples, amouse, trackball, track pad, joystick, game controller, or stylus. Insome embodiments, the input device is the display screen, which is atouch screen or a multi-touch screen. In other embodiments, the inputdevice is a microphone to capture voice or other sound input. In otherembodiments, the input device is a video camera to capture motion orvisual input. In still further embodiments, the input device is acombination of devices such as those disclosed herein. In someembodiments, the systems, and software modules disclosed herein areintranet-based. In some embodiments, the systems and software modulesare Internet-based. In some embodiments, the computer system comprisesWiFi or Bluetooth interfaces. In further embodiments, the computersystems and software modules are World Wide Web-based. In still furtherembodiments, the computer systems and software modules are cloudcomputing-based. In other embodiments, the computer systems and softwaremodules are based on data storage devices including, by way ofnon-limiting examples, CD-ROMs, DVDs, flash memory devices, RAM (e.g.,DRAM, SRAM, etc.), ROM (e.g., PROM, EPROM, EEPROM, etc.), magnetic tapedrives, magnetic disk drives, optical disk drives, magneto-opticaldrives, solid-state drives, and combinations thereof.

In some embodiments, controls systems disclosed herein comprise arobotic arm operably connected to the computer system. In someembodiments, the robotic arm controls a position of the firstbioprinter. In some embodiments the robotic arm moves the bioprinteralong an X, Y, or Z axis, or a combination thereof. In some embodiments,the robotic arm rotates the bioprinter around the X, Y, or Z axis, or acombination hereof. In some embodiments, the robotic arm comprises partof a robotic surgical system. In some embodiments, the robotic surgicalsystem is a Mako robotic surgical system. In some embodiments, therobotic arm is coupled to a body part. In some embodiments, the bodypart is the body part with the internal tissue defect. Coupling to abody part allows the control system to move with the body part shouldthe body part be repositioned. In some embodiments, the control systemcomprises a second robotic arm operatively connected to the computersystem. In some embodiments, the robotic arm controls a position of thesecond bioprinter (FIG. 6 ).

In some embodiments, the control system is configured to control abio-ink printing parameter. In some embodiments the bio-ink printingparameter comprises temperature, backpressure, drops per nozzle,frequency of drop rate, number of nozzles in use, firing energy,resolution, viscosity, cell concentration, physiological temperature,speed of printing, or a combination thereof.

In some embodiments, the systems disclosed herein are capable ofbioprinting a bio-ink construct on an internal tissue defect during aminimally invasive surgery on an individual in need thereof. The systemsmay comprise a control system, an endoscope, a display screen, a sensor,a light source, a 3D scanner, a robotic arm, and a bioprinter comprisinga printhead.

In some embodiments, the systems disclosed herein are portable. In someembodiments, the systems are configured to be moved in and out of anoperating room. By way of non-limiting example, the system may bemounted on wheels or a cart. Also by way of non-limiting example, thesystem may be small and light enough to be carried by hand of an averageuser.

In some embodiments, systems disclosed herein do not require theservices of a live healthcare provider. For example, in someembodiments, the control systems described herein include anon-communication mode. In further embodiments, the methods describedherein are operated in a non-communication mode when communicationprotocols fail, when communication channels or signals fail or are lost,or when devices are placed in a location where one or more communicationprotocols, channels, or signals are unavailable. In a non-communicationmode, a live, remote healthcare provider is unable to monitor,supervise, or operate components of the system. By way of furtherexample, in some embodiments, control systems described herein includean emergency mode. In an emergency mode, in some embodiments, componentsof a system act autonomously, without monitoring, supervision, oroperation by a live or remote healthcare provider.

In some embodiments, the systems are programmable. In some embodiments,the control system is programmable. In some embodiments, the computersystem is programmable. In some embodiments, the control system isprogrammable with information about an internal tissue defect or asubject to be treated using the system. Non-limiting examples ofinformation about the internal tissue defect are shape, size dimension,thickness, density or proximity of the internal tissue defect.

In some embodiments, a healthcare provider programs information aboutthe internal tissue defect into the computer system. In someembodiments, a healthcare provider inputs information about the internaltissue defect into the computer. In some embodiments, the informationabout the internal tissue defect comprises shape, size dimension,thickness, density or proximity of the internal tissue defect. In someembodiments, the, systems disclosed herein are employed, in part or inwhole, in healthcare facilities such as hospitals, hospice, nursinghomes, urgent care offices, diagnostic laboratories, and the like. Insome embodiments, the systems are employed, in part or in whole, inveterinary facilities such as animal hospitals, veterinary offices, andthe like.

In some embodiments, the control system comprises controlling a bio-inkprinting parameter. In some embodiments the bio-ink printing parametercomprises temperature, backpressure, drops per nozzle, frequency of droprate, number of nozzles in use, firing energy, resolution, viscosity,cell concentration, physiological temperature, speed of printing, or acombination thereof.

Display Screens

In some embodiments, the systems disclosed herein comprise a displayscreen. In some embodiments, the system for bioprinting a bio-inkconstruct comprises a plurality of display screens. In some embodiments,the display screen is operatively connected to the computer system. Insome embodiments, the system for bioprinting a bio-ink constructcomprises a connection to a display screen. In some embodiments, thedisplay screen displays an image generated by an endoscope. In someembodiments, the display screen displays an image of an internalstructure of a joint. In some embodiments, the display screen displaysan image of an internal structure of a joint of a patient undergoingminimally invasive surgery. In some embodiments, the display screendisplays an image on an internal structure of a knee of a patientundergoing minimally invasive surgery. In some embodiments, the displayscreen displays an image of a bio-construct printed by the bioprinter.In some embodiments, the display screen displays an image of abio-construct printed by the bioprinter during minimally invasivesurgery. In some embodiments, the display screen displays a real timeimage of a bio-construct being printed by the bioprinter. In someembodiments, the display screen displays a real time image of abio-construct being printed by the bioprinter during minimally invasivesurgery. In some embodiments, the display screen provides visualfeedback of the bioprinting process. In some embodiments, the displayscreen provides visual feedback of the structural accuracy of thebio-construct during the bioprinting process and after the bioprinterprocess. In some embodiments, the display screen provides real timevisual feedback of the location of the bio-construct being bioprinted bythe bioprinter.

In some embodiments, the computer systems described herein include userinterfaces. In further embodiments, the user interfaces include graphicuser interfaces (GUIs). In still further embodiments, the userinterfaces are interactive and present a user with menus and options forinteracting with the computer systems and bioprinters described herein.In further embodiments, the computer system includes a display screen tosend visual information to a user. In some embodiments, the display is acathode ray tube (CRT). In some embodiments, the display screen is aliquid crystal display (LCD). In further embodiments, the display is athin film transistor liquid crystal display (TFT-LCD). In someembodiments, the display is an organic light emitting diode (OLED)display. In various further embodiments, on OLED display is apassive-matrix OLED (PMOLED) or active-matrix OLED (AMOLED) display. Insome embodiments, the display is a plasma display. In other embodiments,the display is a video projector. In some embodiments, the displayscreen is a mobile device screen, a computer screen, a portable devicescreen, a touch screen, or a multi-touch screen. In still furtherembodiments, the display is a combination of displays such as thosedisclosed herein.

Robotic Arms

In some embodiments, the systems disclosed herein comprise at least onerobotic arm. In some embodiments, the controls system comprises arobotic arm operatively connected to the computer system. In someembodiments, the control system comprises a second robotic armoperatively connected to the computer system. In some embodiments, thecontrol system controls the robotic arm. In some embodiments, therobotic arm controls a position of the second bioprinter (FIG. 6 ). Insome embodiments, the printhead is attached distally, with respect tothe user (i.e. a surgeon), to a robotic arm. In some embodiments, aplurality of printheads is attached distally, with respect to the user(i.e. a surgeon), to a plurality of robotic arms. In some embodiments,the printhead is mounted to the distal end, with respect to the user, ofa robotic arm. In some embodiments, a plurality of printheads is mountedto a distal end of a plurality of robotic arms. In some embodiments, theuser is able to manually change or control the depth at which theprinthead, which is attached to a robotic arm, is inserted into apatient during surgery. In some embodiments, the control systemautomatically controls the depth at which the printhead, which isattached to a robotic arm, is inserted into a patient during surgery. Insome embodiments, the distal end of the robotic arm is the end portionof the robotic arm that is closest to the patient.

In some embodiments, a system comprising a plurality of robotic armsdecreases surgery time. In some embodiments, a system comprises aplurality of robotic arms in which one or more arms control one or moreprintheads, while one or more arms control an imaging or a visualizationdevice component or one or more sensors. In some embodiments, a systemcomprising a plurality of printheads decreases surgery time. In someembodiments, a system comprising a plurality of bioprinters decreasessurgery time. In some embodiments, a system comprising a plurality ofrobotic arms enables more than one bio-ink to be deposited. In someembodiments, a system comprising a plurality of printheads enables morethan one bio-ink to be deposited. In some embodiments, a systemcomprising a plurality of bioprinters enables more than one bio-ink tobe deposited. In some embodiments, a system comprising a plurality ofrobotic arms enables a bio-ink construct comprising more than onebio-ink compositions to be bioprinted. In some embodiments, a systemcomprising a plurality of printheads enables a bio-ink constructcomprising more than one bio-ink compositions to be bioprinted. In someembodiments, a system comprising a plurality of bioprinters enables abio-ink construct comprising more than one bio-ink compositions to bebioprinted.

In some embodiments, the robotic arm comprises a base. In someembodiments, the base rotates 360 degrees. In some embodiments, therobotic arm extends from a base. In some embodiments, the robotic armcomprises a joint. In some embodiments, the robotic arm comprises aplurality of joints. In some embodiments, the robotic arm comprises onejoint. In some embodiments, the robotic arm comprises two joints. Insome embodiments, the robotic arm comprises three joints. In someembodiments, the robotic arm comprises four joints. In some embodiments,the robotic arm comprises five joints. In some embodiments, the roboticarm comprises six joints. In some embodiments, the plurality of jointsallows the robotic arm to be rotated about six axes. In someembodiments, the plurality of joints allows the robotic arm to berotated about five axes. In some embodiments, the plurality of jointsallows the robotic arm to be rotated about four axes. In someembodiments, the plurality of joints allows the robotic arm to berotated about three axes. In some embodiments, the robotic arm ismanually rotated by the surgeon. In some embodiments, the robotic arm isautomatically rotated by the control system.

In some embodiments, the robotic arm comprises an actuator. In someembodiments, the actuator is a rotational actuator. In some embodiments,the actuator is an electrical actuator. In some embodiments, theactuator is a direct current (DC) linear actuator. In some embodiments,the actuator is powered by a motor. In some embodiments, the robotic armcomprises a motor or a plurality of motors that power movement of therobotic arm. In some embodiments, the motor is an alternating current(A/C) motor, a direct current (DC) motor, a geared DC motor, an RC servomotor, a stepper motor, or an industrial servo motor. In someembodiments, the motor is controlled by a motor controller. In someembodiments, the motor controller is a brushed DC motor controller, abrushless DC motor controller, a servo motor controller, or a steppermotor controller. In some embodiments, the motor controller isoperatively connected to the control system. In some embodiments, therobotic arm is controlled by the control system. In some embodiments,the motor controller is operatively connected to the computing system.In some embodiments, the robotic arm is controlled by the computingsystem. In some embodiments, the motor controller is operativelyconnected to the computing device. In some embodiments, the robotic armis controlled by the computing device. In some embodiments, the motorcontroller is operatively connected to the microcontroller. In someembodiments, the robotic arm is controlled by the microcontroller. Insome embodiments, the robotic arm is manually controlled. In someembodiments, the robotic arm is controlled by user. In some embodiments,the user is able to manually control the robotic arm at any time duringa surgery. In some embodiments, the user is able to manually change orcontrol the position of the robotic arm at any time during a surgery.

In some embodiments, the robotic arm controls a position of thebioprinter. In some embodiments the robotic arm moves the bioprinteralong an X, Y, or Z axis, or a combination thereof. In some embodiments,the robotic arm rotates the bioprinter around the X, Y, or Z axis, or acombination hereof. In some embodiments, the robotic arm is able to movein any direction and in any angle. In some embodiments, the robotic armhas six degrees of freedom. In some embodiments, the robotic arm hasfive degrees of freedom. In some embodiments, the robotic arm has fourdegrees of freedom. In some embodiments, the robotic arm has threedegrees of freedom. In some embodiments, the robotic arm has two degreesof freedom. In some embodiments, the robotic arm has one degree offreedom. In some embodiments, the robotic arm positions the printheadwithin proximity of or in contact with curved or complex surfaces. Insome embodiments, the robotic arm positions the printhead withinproximity of or in contact with the internal tissue defect. In someembodiments, the robotic arm positions the printhead within proximity ofor in contact with the chondral defect.

In some embodiments, the robotic arm enables the bioprinter to bepositioned at any angle with respect to the substrate being printed on.In some embodiments, the increased flexibility in movement of therobotic arm enables the bioprinter to print directly onto a tissuedefect of a patient. In some embodiments, the robotic arm comprises partof a robotic surgical system. In some embodiments, the robotic surgicalsystem is a Mako robotic surgical system. In some embodiments, therobotic arm is coupled to a body part. In some embodiments, the bodypart is the body part with the internal tissue defect. Coupling to abody part allows the control system to move with the body part shouldthe body part be repositioned.

Light Sources

In some embodiments, the systems disclosed herein comprise a lightsource. In some embodiments, the light source is positioned withinproximity of the internal tissue defect. In some embodiments, the lightsource is a laser. In some embodiments, the light source is a lamp. Insome embodiments, the light source emits light in a focused region. Insome embodiments, the light source emits light in a pattern. In someembodiments, the pattern of light cross-links the bio-ink. In someembodiments, the light source polymerizes the bio-ink. In someembodiments, the method comprises a wash step to remove the bio-inkwhich was not cross-linked. In some embodiments, the light source isconnected to the endoscope. In some embodiments, the light source emitsUV light. In some embodiments, UV light comprises UV-A light, UV-Blight, or UV-C light. In some embodiments, UV-A light comprises awavelength of light between about 315 nm and about 400 nm. In someembodiments, UV-B light comprises a wavelength of light between about280 nm and about 315 nm. In some embodiment, UV-C light comprises awavelength of light between about 100 nm to about 280 nm. In someembodiments, the light source is a light emitting diode (LED).

In some embodiments, the system comprises a light source that excitesthe bio-ink. In some embodiments, the system comprises a light sourcethat excites a cell in the bio-ink. In some embodiments, the systemcomprises a light source that excites one or more components of thebio-ink. In some embodiments, one or more components of the bio-ink aredetectable. In some embodiments, the components of the bio-ink, that areexcited by the light source, comprise a plurality of cells, a protein, acell adhesion molecule, an extracellular matrix component, a growthfactor, an enzyme, a therapeutic agent, a natural polymer, a syntheticpolymer, a hydrogel, a nanoparticle, a biochemical factor, across-linking agent, a photoinitiator, an additional agent, or acombination thereof. In some embodiments, the plurality of cells excitedby the light source is labeled with a fluorescent probe, a fluorescenttag, an affinity tag, or a peptide. In some embodiments, the pluralityof cells excited by the light source is a plurality of: chondrocytes,chondrogenic precursors, chondroprogenitors, stem cells, pluripotentstem cells, induced pluripotent stem cells, mesenchymal stem cells, orany combination thereof. In some embodiments, the system comprises alight source to excite a protein in the bio-ink. In some embodiments,the protein excited by the light source is labeled with a fluorescentprobe, a fluorescent tag, an affinity tag, or a peptide. In someembodiments, the protein excited by the light source autofluoresces. Insome embodiments, the autofluorescent protein that is excited by thelight source is collagen.

In some embodiments, the light source emits light with a visiblewavelength of to 400 nm to 700 nm. In some embodiments, the light sourceemits light at a wavelength between about 270 nm to about 370 nm. Insome embodiments, the light source emits light at a wavelength betweenabout 480 nm to about 500 nm. In some embodiments, the light sourceemits light at a wavelength of about 488 nm. In some embodiments, thelight source emits light at a wavelength between about 540 nm to about570 nm. In some embodiments, the light source emits light at awavelength of about 561 nm. In some embodiments, the light source emitslight at a wavelength of about 546 nm. In some embodiments, the lightsource emits light at a wavelength between about 620 nm to about 640 nm.In some embodiments, the light source emits light at a wavelength ofabout 633 nm. In some embodiments, the light source emits light at awavelength between about 640 nm to about 660 nm. In some embodiments,the light source emits light at a wavelength of about 647 nm.

Sensors

In some embodiments, the systems disclosed herein comprise a sensor. Insome embodiments, the systems disclosed herein comprise a plurality ofsensors. In some embodiments, the sensor is configured to detect andmonitor the position of the robotic arm. In some embodiments, the sensoris configured to detect and monitor the position of the bioprinter. Insome embodiments, the sensor is configured to detect and monitor theposition of the printhead. In some embodiments, the sensor is configuredto detect and monitor the position of a nozzle or a plurality ofnozzles. In some embodiments, the sensor is configured to detect andmonitor the position of a needle. In some embodiments, the sensor is anoptical sensor, a rotary encoder, a piezoelectric accelerometer, acapacitive displacement sensor, a gyroscopic sensor, a pressure sensor,an infrared sensor, a linear potentiometer, a stretch sensor, a stereocamera system, a localization system, a light sensor, a thermal sensor,a temperature sensor, a thermal camera, an inertial measurement unit(IMU), a current sensor, a voltage sensor, a magnetic sensor, anelectromagnetic sensor, a depth sensor, an acoustic sensor, a touchsensor, a confocal displacement sensor, or any combination thereof.

In some embodiments, the sensor detects the temperature of the bio-ink.In some embodiments, the sensor detects the volume of bio-ink beingejected from the bioprinter. In some embodiments, the sensor detects thedegree of rotation of a joint in the robotic arm. In some embodiments,the sensor detects the translation of the robotic arm. In someembodiments, the sensor detects a distance from the bioprinter to thesubstrate. In some embodiments, the sensor detects a distance from thebioprinter to the tissue defect. In some embodiments, the sensor detectsa distance within a substrate. In some embodiments, the sensor detects adistance within a tissue defect. In some embodiments, the sensorprovides location feedback to the control system. In some embodiments,the sensor measures a depth of the bio-ink construct. In someembodiments, the sensor measures a depth of the tissue defect. In someembodiments, the sensor measures a temperature of the bio-ink or of thebio-ink construct. In some embodiments, the sensor measures atemperature of the tissue defect.

Endoscopes

In some embodiments, the systems disclosed herein comprise at least oneendoscope. In some embodiments, the endoscope is positioned withinproximity of the internal tissue defect. In some embodiments, theendoscope visualizes the internal tissue defect during bioprinting. Insome embodiments, the endoscope is an arthroscope, bronchoscope,colonoscope, colposcope, cystoeurethroscope, cystoscope, duodenoscope,enteroscope, esophagogastroduodenoscope, fetoscope, gastroscope,gynoscope, hysteroscope, laparoscope, laryngoscope, peritoneoscope,proctosigmoidoscope, sigmoidoscope, thoracoscope, or ureteroscope. Insome embodiments, the system comprises a first endoscope, a secondendoscope, a third endoscope, a fourth endoscope, or a fifth endoscopewithin proximity of the internal tissue defect.

In some embodiments, the system comprises an endoscope to detect orvisualize the bio-ink during or after bio-printing. In some embodiments,the system comprises an endoscope configured to provide an image of theinternal tissue defect, wherein the image is used to provide feedbackregarding the structure of a bio-ink construct during a bio-printingprocess in real time. In some embodiments, the endoscope providesfeedback regarding the structure of the bio-construct during thebio-printing process in real time. In some embodiments, the systemcomprises an endoscope to detect or visualize one or more components ofthe bio-ink. In some embodiments, the components of the bio-ink comprisea plurality of cells, a protein, a cell adhesion molecule, anextracellular matrix component, a growth factor, an enzyme, atherapeutic agent, a natural polymer, a synthetic polymer, a hydrogel, ananoparticle, a biochemical factor, a cross-linking agent, aphotoinitiator, an additional agent, or a combination thereof. In someembodiments, the system comprises an endoscope to detect or visualize aplurality of cells in the bio-ink. In some embodiments, the plurality ofcells detected or visualized by the endoscope is labeled with afluorescent probe, a fluorescent tag, an affinity tag, or a peptide. Insome embodiments, the plurality of cells detected or visualized by theendoscope is a plurality of: chondrocytes, chondrogenic precursors,chondroprogenitors, stem cells, pluripotent stem cells, inducedpluripotent stem cells, mesenchymal stem cells, or any combinationthereof.

In some embodiments, the system comprises an endoscope to detect orvisualize a protein in the bio-ink. In some embodiments, the proteindetected or visualized by the endoscope is labeled with a fluorescentprobe, a fluorescent tag, an affinity tag, or a peptide. In someembodiments, the protein detected or visualized by the second endoscopeautofluoresces. In some embodiments, the autofluorescent proteindetected or visualized by the endoscope is collagen. In someembodiments, the autofluorescent collagen comprises collagen type I,collagen type IL collagen type Ill, collagen type IV, collagen type V,collagen type VI, collagen type VII, collagen type VIII, collagen typeIX, collagen type X, collagen type XI, collagen type XII, collagen typeXIII, collagen type XIV, collagen type XV, collagen type XVI, collagentype XVII, collagen type XVIII, collagen type XIX, collagen type XX,collagen type XXI, collagen type XXII, collagen type XXIII, collagentype XXIV, collagen type XXV, collagen type XXVI, collagen type XXVII,collagen type XXVIII, collagen type XXIX or a combination thereof.

In some embodiments, the system comprises at least a first endoscope anda second endoscope, wherein the first endoscope detects or visualizes afirst bio-ink during or after bio-printing, and the second endoscopedetects or visualizes a second bio-ink during or after bio-printing. Insome embodiments, the first bio-ink is different from the secondbio-ink. In some embodiments, the first bio-ink and the second bio-inkare the same. In some embodiments, the system comprises at least a firstendoscope and a second endoscope, wherein the first endoscope detects orvisualizes a first component of a first bio-ink during or afterbio-printing, and the second endoscope detects or visualizes a secondcomponent of a second bio-ink during or after bio-printing. In someembodiments, the system comprises at least a first endoscope and asecond endoscope, wherein the first endoscope detects or visualizes afirst component of a first bio-ink during or after bio-printing, and thesecond endoscope detects or visualizes a second component of the firstbio-ink during or after bio-printing. In some embodiments, the firstcomponent and the second component are the same. In some embodiments,the first component and the second component are different. In someembodiments, there are additional endoscopes (e.g., third, fourth, fifthendoscope) that detect or visualize additional bio-inks or additionalcomponents of bio-inks. 3D Scanners

In some embodiments, the systems disclosed herein comprise a threedimensional (3D) scanner. In some embodiments, the 3D scanner is a threedimensional laser scanner. In some embodiments, the 3D scanner is ahand-held laser scanner. In some embodiments, the 3D scanner is a threedimensional X-ray scanner. In some embodiments, the 3D scanner is astructured light three dimensional. In some embodiments, the 3D scanneris a contact 3D scanner. In some embodiments, the 3D scanner is amodulated light 3D scanner. In some embodiments, the 3D scanner is astationary 3D laser scanner. In some embodiments, the 3D scanner is atime-of-flight 3D scanner. In some embodiments, the time-of-flight is alaser phase-shift 3D scanner or a laser pulsed-based 3D scanner.

In some embodiments, the 3D scanner is located in a flexible, tubularinstrument, similar in structure to an endoscope. In some embodiments,the 3D scanner located in close proximity to or in contact with a tissuedefect. In some embodiments, the 3D scanner is located in closeproximity to or in contact with an osteochondral defect. In someembodiments, the 3D scanner is located in close proximity to or incontact with a chondral defect.

In some embodiments, the 3D scanner scans the internal tissue defect. Insome embodiments, the 3D scanner scans the bio-ink. In some embodiments,the 3D scanner scans the bio-ink construct. In some embodiments, the 3Dscanner creates a point cloud of the internal tissue defect. In someembodiments, a point cloud is a set of data points in a threedimensional coordinate system that is measured and outputted by the 3Dscanner. In some embodiments, the 3D scanner creates a point cloud ofthe internal tissue defect before, during, or after the bioprintingprocess. In some embodiments, the 3D scanner creates a point cloud ofthe bio-ink. In some embodiments, the 3D scanner creates a point cloudof the bio-ink construct. In some embodiments, the 3D scanner creates apoint cloud of the bio-ink or bio-ink construct before, during, or afterthe bioprinting process. In some embodiments, the control system usesthe point cloud created by the 3D scanner to design a bio-ink construct.In some embodiments, the control system uses the point cloud created bythe 3D scanner to direct the bioprinter. In some embodiments, thecontrol system uses the point cloud created by the 3D scanner todetermine the bio-ink printing parameters. In some embodiments, thecontrol system uses the point cloud created by the 3D scanner to providespatial, structural, or geometrical feedback of the bio-ink or bio-inkconstruct. In some embodiments, the spatial, structural, or geometricalfeedback of the bio-ink or bio-ink construct is generated before,during, or after the bioprinting process.

In some embodiments, the 3D scanner provides feedback regarding thestructure of the bio-construct during the bio-printing process in realtime. In some embodiments, the 3D scanner provides a feedback regardingthe location of the bio-construct during the bio-printing process inreal time. In some embodiments, the 3D scanner provides a feedbackregarding the location of the bio-ink during the bio-printing process inreal time. In some embodiments, the feedback provided by the 3D scanneris spatial feedback. In some embodiments, the spatial feedback iscalculated by the computing system. In some embodiments, the computingsystem executes instructions to take a 3D scan of the bio-constructduring the bio-printing process. In some embodiments, the computingsystem executes instructions to take a 3D scan of the substrate ortissue defect during the bio-printing process. In some embodiments, thecomputing system executes instructions to take a 3D scan of thebio-construct during the bio-printing process, compare it to theoriginal design to be printed of the bio-construct structure, and detectany structural differences between them. In some embodiments, thecomputing system executes instructions to take a first 3D scan of thesubstrate or tissue defect, take a second 3D scan of the substrate ortissue defect during the bio-printing process, compare the first andsecond 3D scans, and detect any differences between the first and secondscan.

In some embodiments, the 3D scanner provides an image of the internaltissue defect. In some embodiments, the 3D scanner provides an image ofthe internal tissue defect that is used to design a bio-ink construct.In some embodiments, the 3D scanner provides an image of the internaltissue defect that is used to determine the shape or structure of thebio-ink construct. In some embodiments, the 3D scanner provides an imageof the internal tissue defect that is used to design a bio-ink constructthat fits or aligns with a tissue defect of a patient. In someembodiments, the 3D scanner provides an image of the internal tissuedefect that is used to design a bio-ink construct that is patientspecific. In some embodiments, the 3D scanner provides an image of theinternal tissue defect that is used to design a bio-ink construct thatmatches the shape or construct of the tissue defect of the patient. Insome embodiments, the 3D scanner provides an image of the internaltissue defect that is used to design a bio-ink construct thatcomplements the shape or construct of the tissue defect of the patient.In some embodiments, the 3D scanner provides an image of the internaltissue defect that is used by the control system to determine theinstructions to be sent to the bioprinter. In some embodiments, the 3Dscanner provides an image of the internal tissue defect that is used bythe control system to determine the bio-ink printing parameters.

In some embodiments, the 3D scanner provides a point cloud of theinternal tissue defect. In some embodiments, the 3D scanner provides apoint cloud of the internal tissue defect that is used to design abio-ink construct. In some embodiments, the 3D scanner provides a pointcloud of the internal tissue defect that is used to determine the shapeor structure of the bio-ink construct. In some embodiments, the 3Dscanner provides a point cloud of the internal tissue defect that isused to design a bio-ink construct that fits or aligns with a tissuedefect of a patient. In some embodiments, the 3D scanner provides apoint cloud of the internal tissue defect that is used to design abio-ink construct that is patient specific. In some embodiments, the 3Dscanner provides a point cloud of the internal tissue defect that isused to design a bio-ink construct that matches the shape or constructof the tissue defect of the patient. In some embodiments, the 3D scannerprovides a point cloud of the internal tissue defect that is used todesign a bio-ink construct that complements the shape or construct ofthe tissue defect of the patient. In some embodiments, the 3D scannerprovides a point cloud of the internal tissue defect that is used by thecontrol system to determine the instructions to be sent to thebioprinter. In some embodiments, the 3D scanner provides a point cloudof the internal tissue defect that is used by the control system todetermine the bio-ink printing parameters.

Uses of Compositions and Methods

Provided herein are compositions and methods for treating a subject inneed thereof. In some embodiments, the subject is a burn victim, anathlete or an amputee. In some embodiments, the subject is an elderlyindividual, an infant, or a youth. In some embodiments, the subject isin need of an organ transplant. In some embodiments, the subject in needof an organ transplant requires an eye, a heart, a lung, a stomach, anintestine, a colon, a bladder, a pancreas, a spleen, a uterus, an ovary,a prostate, a muscle, a bone, an artery, a blood vessel, a thyroid, aliver or a kidney. In some embodiments, the subject suffers from anautoimmune disease, a cardiovascular disorder, an autophageal disorderor a neurodegenerative disorder. In some embodiments, theneurodegenerative disorder is selected from Parkinson’s Disease,Alzheimer’s Disease, Huntington’s Disease, Amyotrophic Lateral Sclerosis(Lou Gehrig’s Disease), or macular degeneration. In some embodiments,the autoimmune disease is selected from multiple sclerosis,encephalomyelitis, hepatitis, inner ear disease, peripheral neuropathyor pancreatitis. In some embodiments, the subject is suffering frombrain trauma, tissue degeneration, cancer, arthritis, osteoarthritis,gout, tooth decay or an ulcer. In some embodiments, subject suffers fromosteoarthritis. In some embodiments, the subject is a human. In someembodiments, the subject is an animal. In further embodiments, theanimal is under the care of an owner, caretaker, rescuer, orveterinarian. In still further embodiments, the animal is aninvertebrate, fish, amphibian, reptile, bird, or mammal.

Provided herein are compositions and methods for treating an internaldefect of a subject in need thereof. In some embodiments, the internaltissue defect is located in or on a tissue or organ selected from avascular tissue, an osteochondral tissue, an epidermal tissue, amuscular tissue, an intestinal tissue, a neuronal tissue, a reproductivetissue, a pancreatic tissue, an ocular tissue, an ear, an eye, a cornea,a nose, a brain, a sinus, a tooth, a bone, cartilage, skin, anesophagus, a trachea, a thymus, a thyroid, a heart, a blood vessel, alung, a diaphragm, a lymph node, a breast, a nipple, a stomach, anintestine, a colon, a rectum, a pancreas, a spleen, a bladder, a kidney,a liver, an ovary, a uterus, a vagina, a prostate, a penis, a cervix,adipose, skeletal muscle, smooth muscle or skin. In some embodiments,the skin is located on a head, on a face, on a neck, on a shoulder, on achest, on an arm, on a hand, on a back, on a buttock, on a leg, on anankle or on a foot. In some embodiments, the method comprises treatingan internal tissue defect in a joint. In some embodiments, the j oint islocated in a neck, a shoulder, a back, a spine, a chest, an arm, a hand,an elbow, a wrist, a finger, a leg, an ankle, a foot, a hip or a knee.In some embodiments, the joint is located in a knee.

In some embodiments, the internal tissue defect is a birth defect or acongenital defect. In some embodiments, the internal tissue defect is aresult of an injury. In some embodiments, the injury is due tomusculoskeletal trauma, a sport injury, an automobile accident, aninfection, or a tumor. In some embodiments, the method comprisestreating an internal tissue defect wherein the internal tissue defect isselected from a damaged tissue, eroded tissue, diseased tissue ordegenerated tissue. In some embodiments, the damaged tissue is selectedfrom a tissue damaged by a burn, an abrasion, a tear, a lesion, a break,a fracture, a bruise, a hematoma, a scratch, a cut, a puncture, aninfection, a tumor, frostbite, overuse or necrosis. In some embodiments,the method comprises characterization of the internal tissue defect. Insome embodiments, the method comprises x-ray, CAT/CT scan, PET scan,MRI, ultrasound, thermography, endoscopy, radiography or biopsy of theinternal tissue defect. In some embodiments, the method comprisespreparation of the internal tissue defect before surgery. In someembodiments, preparation of the internal tissue defect comprises tissueremoval, radiation, sterilization, cleaning, treatment with anantibiotic or treatment with an anesthetic.

In some embodiments, the tissue defect is an articular joint defect. Insome embodiments, the tissue defect is a chondral defect. In someembodiments, a chondral defect is a focal area of injury or damage toarticular cartilage. In some embodiments, the tissue defect is anosteochondral defect. In some embodiments, an osteochondral defect is afocal area of injury or damage to articular cartilage in combinationwith damage or injury to adjacent subchondral bone. In some embodiments,the tissue defect is a tissue defect in the knee, a tissue defect in thehip, a tissue defect in the shoulder, a tissue defect in the ankle, or atissue defect in the elbow. In some embodiments, the tissue defect is afractured bone. In some embodiments, the tissue defect is a fracture inthe knee, a fracture in the hip, a fracture in the shoulder, a fracturein the ankle, or a fracture in the elbow. In some embodiments, thetissue defect is a torn ligament. In some embodiments, the tissue defectis a torn ligament in the knee, a torn ligament in the hip, a tornligament in the shoulder, a torn ligament in the ankle, or a tornligament in the elbow. In some embodiments, the tissue defect is a torntendon. In some embodiments, the tissue defect is a torn tendon in theknee, a torn tendon in the hip, a torn tendon in the shoulder, a torntendon in the ankle, or a torn tendon in the elbow. In some embodiments,the tissue defect is a swollen synovium. In some embodiments, the tissuedefect is a torn articular cartilage. In some embodiments, the tissuedefect is a meniscal cartilage tear. In some embodiments, the tissuedefect is a surface cartilage tear. In some embodiments, the tissuedefect is a torn meniscus. In some embodiments, the tissue defect is aknee bone fracture. In some embodiments, the tissue defect is a tornanterior cruciate ligament or a torn posterior cruciate ligament. Insome embodiments, the tissue defect is a Baker’s cyst. In someembodiments, the tissue defect is a tear on the labrum.

The disclosure is further understood through review of the numberedembodiments recited herein. Various embodiments contemplated herein mayinclude, but need not be limited to, one or more of the following, andcombinations thereof:

-   1. A method of bioprinting a bio-ink construct on an internal tissue    defect during a minimally invasive surgery on an individual in need    thereof, comprising: a. visualizing the internal tissue defect; b.    positioning a bioprinter comprising a printhead within proximity of    or in contact with the internal tissue defect; and c. ejecting a    bio-ink from the printhead onto the internal tissue defect to form a    bio-ink layer, thereby generating a bio-ink construct. 2. The method    of claim 1, wherein the bio-ink construct comprises a plurality of    bio-ink layers. 3. The method of claim 1, wherein the bio-ink    construct is a live tissue. 4. The method of claim 1, wherein the    printhead comprises a needle, an extended cylinder, a fluid line, a    print nozzle, or a plurality of print nozzles. 5. The method of    claim 4, wherein each print nozzle of the plurality of print nozzles    is independently controlled and actuated. 6. The method of claim 4,    wherein each print nozzle of the plurality of print nozzles is    actuated to eject an individual droplet of the bio-ink. 7. The    method of claim 6, wherein the plurality of print nozzles ejects the    individual droplet simultaneously. 8. The method of claim 6, wherein    the plurality of print nozzles ejects the individual droplet in a    specified sequence. 9. The method of claim 1, wherein the printhead    ejects the bio-ink continuously. 10. The method of claim 1, wherein    the bio-ink comprises a plurality of cells, a component of    extracellular matrix, a synthetic polymer, a natural polymer, a    cross-linking agent, a photoinitiator, or a combination thereof. 11.    The method of claim 10, wherein the plurality of cells comprises    cells selected from chondrocytes, connective tissue fibroblasts,    tendon fibroblasts, bone marrow reticular tissue fibroblasts,    non-epithelial fibroblasts, pericytes, osteoprogenitor cells,    osteoblasts, osteoclasts, keratinocytes, hair root cells, hair shaft    cells, hair matrix cells, exocrine secretory epithelial cells,    hormone secreting cells, epithelial cells, neural or sensory cells,    photoreceptor cells, muscle cells, extracellular matrix cells, blood    cells, cardiovascular cells, endothelial cells, kidney cells,    hepatic cells, pancreatic cells, immune cells, stem cells, germ    cells, nurse cells, interstitial cells, stellate cells and    progenitors thereof. 12. The method of claim 10, wherein the    plurality of cells comprises cells selected from chondrocytes,    connective tissue fibroblasts, tendon fibroblasts, bone marrow    reticular tissue fibroblasts, non-epithelial fibroblasts, pericytes,    osteoprogenitor cells, osteoblasts, osteoclasts, and progenitors    thereof. 13. The method of claim 10, wherein the plurality of cells    comprises chondrocytes. 14. The method of claim 10, wherein the    component of extracellular matrix comprises collagen, elastin,    fibrillin, fibronectin, laminin, fibrinogen, tenascin,    thrombospondin, integrin, hyaluronic acid, heparin, heparin sulfate,    chondroitin sulfate, keratin sulfate, dermatan sulfate, or a    combination thereof. 15. The method of claim 10, wherein the    synthetic polymer is polyethylene glycol (PEG), a PEG macromere,    polyethylene glycol methacrylate (PEGMA), polyethylene    dimethacrylate (PEDGMA), poly(hydroxyethyl methacrylate) (PHEMA),    polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), carboxymethyl    cellulose (CMC), polyimide (PI), polyacrylate (PAA), polyurethane    (PU), PEG-lactide, PEG-glycolide, or a combination thereof. 16. The    method of claim 10, wherein the natural polymer is alginate,    cellulose, gelatin, pectin, agarose, chitosan, or a combination    thereof. 17. The method of claim 10, wherein the cross-linking agent    comprises calcium chloride, calcium sulfate, calcium carbonate,    calcium (Ca2+), magnesium (Mg2+), glutaraldehyde, genipin,    nordihydroguaiaretic acid, tannin acid, procyanidins,    glycosaminoglycan (GAG), 1-ethyl-3-3-dimethylaminopropylcarbodiimide    hydrochloride (EDC), divinyl benzene (DVB), ethylene glycol    dimethacrylate (EGDMA), tetraethylene glycol diacrylate (TEGDA),    polyethylene glycol diacrylate (PEGDA), or a combination    thereof. 18. The method of claim 1, further comprising polymerizing    the bio-ink. 19. The method of claim 18, wherein polymerizing the    bio-ink comprises cross-linking the bio-ink. 20. The method of claim    19, wherein cross-linking the bio-ink comprises delivering the    cross-linking agent by the printhead to the bio-ink. 21. The method    of claim 19, wherein cross-linking the bio-ink comprises applying UV    light from a light source to the bio-ink. 22. The method of claim    19, wherein cross-linking the bio-ink comprises applying heat to the    bio-ink. 23. The method of claim 1, wherein the bioprinter comprises    a second printhead. 24. The method of claim 1, further comprising    positioning a second bioprinter comprising a printhead within    proximity of or in contact with the internal tissue defect. 25. The    method of claim 24, further comprising ejecting a second bio-ink    from the printhead of the second bioprinter onto the internal tissue    defect to form a second bio-ink layer. 26. The method of claim 1,    further comprising controlling the bioprinter with a control    system. 27. The method of claim 26, wherein the control system    comprises a computer system. 28 The method of claim 27, wherein the    control system comprises a robotic arm operatively connected to the    computer system. 29. The method of claim 28, wherein the robotic arm    is coupled to a body part of the individual. 30. The method of claim    28, wherein the robotic arm positions the bioprinter. 31. The method    of claim 30, wherein the bioprinter is moved along an X, Y, or Z    axis, or a combination thereof. 32. The method of claim 30, wherein    the bioprinter is rotated around the X, Y, or Z axis, or a    combination thereof. 33. The method of claim 26, wherein the control    system controls a bio-ink printing parameter. 34. The method of    claim 33, wherein the bio-ink printing parameter comprises    temperature, back-pressure, drops per nozzle, frequency of drop    rate, number of nozzles in use, firing energy, resolution,    viscosity, cell concentration, physiological temperature, speed of    printing, or a combination thereof. 35. The method of claim 1,    wherein visualizing the internal tissue defect occurs before,    during, or after ejecting the bio-ink. 36. The method of claim 1,    wherein visualizing the internal tissue defect comprises imaging the    internal tissue defect. 37. The method of claim 1, further    comprising positioning an endoscope within proximity of the internal    tissue defect. 38. The method of claim 37, wherein the endoscope    visualizes the internal tissue defect. 39. The method of claim 1,    wherein the internal tissue defect is selected from a damaged    tissue, eroded tissue, diseased tissue or degenerated tissue. 40.    The method of claim 1, wherein the internal tissue defect is in an    internal tissue selected from bone, muscle, nerves, brain, eye,    pancreas, spleen, cartilage, thyroid, adipose, sinus, esophagus,    kidney, heart, lung, intestine, stomach, colon, rectum, breast,    ovary, uterus, cervix, prostate, bladder or liver. 41. The method of    claim 1, wherein the internal tissue defect is selected from a    vascular defect, a chondral defect, a muscular defect, an intestinal    defect, a neuronal defect, a reproductive defect, a pancreatic    defect, or an ocular defect. 42. The method of claim 1, wherein the    internal tissue defect comprises a chondral defect. 43. The method    of claim 42, wherein the chondral defect is in a joint selected from    a knee joint, a hip joint, an elbow joint, a shoulder joint, a wrist    joint, a spine joint, a finger joint, an ankle joint, or a foot    joint. 44. The method of claim 42, wherein the chondral defect is in    a knee joint. 45. The method of claim 42, wherein the chondral    defect is an osteochondral defect. 46. A method of bioprinting a    bio-ink construct on a chondral defect during a minimally invasive    surgery on an individual in need thereof comprising: a. visualizing    the chondral defect; b. positioning a bioprinter comprising a    printhead within proximity of or in contact with the chondral    defect; and c. ejecting a bio-ink from the printhead onto the    chondral defect to form a bio-ink layer, thereby generating a    bio-ink construct. 47. The method of claim 46, wherein the bio-ink    construct comprises a plurality of bio-ink layers. 48. he method of    claim 46, wherein the bio-ink construct is a live tissue. 49. The    method of claim 1, wherein the printhead comprises a needle, an    extended cylinder, a fluid line, a print nozzle, or a plurality of    print nozzles. 50. The method of claim 49, wherein each print nozzle    of the plurality of print nozzles is independently controlled and    actuated. 51. The method of claim 49, wherein each print nozzle of    the plurality of print nozzles is actuated to eject an individual    droplet of the bio-ink. 52. The method of claim 51, wherein the    plurality of print nozzles ejects the individual droplet    simultaneously. 53. The method of claim 51, wherein the plurality of    print nozzles ejects the individual droplet in a specified    sequence. 54. The method of claim 1, wherein the printhead ejects    the bio-ink continuously. 55. The method of claim 46, wherein the    bio-ink comprises a plurality of cells, a component of extracellular    matrix, a synthetic polymer, a natural polymer, a cross-linking    agent, a photoinitiator, or a combination thereof. 56. The method of    claim 55, wherein the plurality of cells comprises cells selected    from chondrocytes, connective tissue fibroblasts, tendon    fibroblasts, bone marrow reticular tissue fibroblasts,    non-epithelial fibroblasts, pericytes, osteoprogenitor cells,    osteoblasts, osteoclasts, and progenitors thereof. 57. The method of    claim 55, wherein the plurality of cells comprises chondrocytes. 58.    The method of claim 55, wherein the component of extracellular    matrix comprises collagen, elastin, fibrillin, fibronectin, laminin,    fibrinogen, tenascin, thrombospondin, integrin, hyaluronic acid,    heparin, heparin sulfate, chondroitin sulfate, keratin sulfate,    dermatan sulfate, or a combination thereof. 59. The method of claim    55, wherein the synthetic polymer is polyethylene glycol (PEG), a    PEG macromere, polyethylene glycol methacrylate (PEGMA),    polyethylene dimethacrylate (PEDGMA), poly(hydroxyethyl    methacrylate) (PHEMA), polyvinyl alcohol (PVA), polyvinylpyrrolidone    (PVP), carboxymethyl cellulose (CMC), polyimide (PI), polyacrylate    (PAA), polyurethane (PU), PEG-lactide, PEG-glycolide, or a    combination thereof. 60. The method of claim 55, wherein the natural    polymer is alginate, cellulose, gelatin, pectin, agarose, chitosan,    or a combination thereof. 61. The method of claim 55, wherein the    cross-linking agent comprises calcium chloride, calcium sulfate,    calcium carbonate, calcium (Ca2+), magnesium (Mg2+), glutaraldehyde,    genipin, nordihydroguaiaretic acid, tannin acid, procyanidins,    glycosaminoglycan (GAG), 1-ethyl-3-3-dimethylaminopropylcarbodiimide    hydrochloride (EDC), divinyl benzene (DVB), ethylene glycol    dimethacrylate (EGDMA), tetraethylene glycol diacrylate (TEGDA),    polyethylene glycol diacrylate (PEGDA), or a combination    thereof. 62. The method of claim 46, further comprising polymerizing    the bio-ink. 63. The method of claim 46, wherein polymerizing the    bio-ink comprises cross-linking the bio-ink. 64. The method of claim    63, wherein cross-linking the bio-ink comprises delivering the    cross-linking agent by the printhead to the bio-ink. 65. The method    of claim 63, wherein cross-linking the bio-ink comprises applying UV    light from a light source to the bio-ink. 66. The method of claim    63, wherein cross-linking the bio-ink comprises applying heat to the    bio-ink. 67. The method of claim 46, wherein the bioprinter    comprises a second printhead. 68. The method of claim 67, further    comprising positioning a second bioprinter comprising a printhead    within proximity of or in contact with the chondral defect. 69. The    method of claim 68, further comprising ejecting a second bio-ink    from the printhead of the second bioprinter onto the chondral defect    to form a second bio-ink layer. 70. The method of claim 46, further    comprising controlling the bioprinter with a control system. 71. The    method of claim 70, wherein the control system comprises a computer    system. 72. The method of claim 71, wherein the control system    comprises a robotic arm operatively connected to the computer    system. 73. The method of claim 72, wherein the robotic arm is    coupled to a body part of the individual. 74. The method of claim    73, wherein the robotic arm positions the bioprinter. 75. The method    of claim 74, wherein the bioprinter is moved along an X, Y, or Z    axis, or a combination thereof. 76. The method of claim 74, wherein    the bioprinter is rotated around the X, Y, or Z axis, or a    combination thereof. 77. The method of claim 71, wherein the control    system controls a bio-ink printing parameter. 78. The method of    claim 77, wherein the bio-ink printing parameter comprises    temperature, back-pressure, drops per nozzle, frequency of drop    rate, number of nozzles in use, firing energy, resolution,    viscosity, cell concentration, physiological temperature, speed of    printing, or a combination thereof. 79. The method of claim 46,    wherein visualizing the chondral defect occurs before, during, or    after ejecting the bio-ink. 80. The method of claim 46, wherein    visualizing the chondral defect comprises imaging the chondral    defect. 81. The method of claim 46, further comprising positioning    an endoscope within proximity of the chondral defect. 82. The method    of claim 81, wherein the endoscope visualizes the chondral    defect. 83. The method of claim 46, wherein the chondral defect is    selected from a damaged tissue, eroded tissue, diseased tissue or    degenerated tissue. 84. The method of claim 46, wherein the chondral    defect is in a joint selected from a knee joint, a hip joint, an    elbow joint, a shoulder joint, a wrist joint, a spine joint, a    finger joint, an ankle joint, or a foot joint. 85. The method of    claim 46, wherein the chondral defect is in a knee joint. 86. The    method of claim 46, wherein the chondral defect is an osteochondral    defect. 87. A system for bioprinting a bio-ink construct on an    internal tissue defect during a minimally invasive surgery on an    individual in need thereof, comprising a control system, an    endoscope, and a bioprinter comprising a printhead. 88. The system    of claim 87, further comprising a light source. 89. The system of    claim 87, wherein the printhead comprises a needle, an extended    cylinder, a fluid line, a print nozzle, or a plurality of print    nozzles. 90. The system of claim 87, wherein the bioprinter    comprises a second printhead. 91. The system of claim 87, further    comprising a second bioprinter. 92. The system of claim 87, wherein    the control system controls the bioprinter. 93. The system of claim    87, wherein the control system comprises a computer system. 94. The    system of claim 93, wherein the control system comprises a robotic    arm operatively connected to the computer system. 95. The system of    claim 94, wherein the robotic arm is coupled to a body part. 96. The    system of claim 94, wherein the robotic arm positions the    bioprinter. 97. The system of claim 96, wherein the bioprinter is    moved along an X, Y, or Z axis, or a combination thereof. 98. The    system of claim 96, wherein the bioprinter is rotated around the X,    Y, or Z axis, or a combination thereof. 99. The system of claim 87,    wherein the control system controls a bio-ink printing    parameter. 100. The system of claim 99, wherein the bio-ink printing    parameter comprises temperature, back-pressure, drops per nozzle,    frequency of drop rate, number of nozzles in use, firing energy,    resolution, viscosity, cell concentration, physiological    temperature, speed of printing, or a combination thereof. 101. The    system of claim 100, wherein the firing energy includes pulse    energy, pulse width, length of gap between pulses, and voltage. 102.    The system of claim 94, wherein the control system comprises a    second robotic arm operatively connected to the computer    system. 103. The system of claim 102, wherein the robotic arm    controls a position of the second bioprinter.

EXAMPLES Example 1: Repairing a Chondral Defect in the Knee DuringSurgery, Using a Single Printhead in A Single Bioprinter

A 16- year old boy has a chondral defect of the knee joint due to arepetitive sports injury. Healthy human articular cartilage is harvestedfrom the boy and is rinsed and sterilized with phosphate buffered saline(PBS). Sterile scalpels are used to excise articular cartilage fromfemoral condyles and tibia plateaus under aseptic conditions. Harvestedcartilage samples arc minced and treated with 0.5 mg/mL trypsin at 37°C. for 15 min. After removing trypsin solution, the cartilage tissuesare digested with 2 mg/mL type IV clostridial collagenase in DMEM with5% fetal calf serum for 12 h to 16 h at 37° C. Released human articularchondrocytes are washed three times with DMEM supplemented with 1Xpenicillin-streptomycin-glutamine (PSG) and cell viability isdetermined. Isolated chondrocytes are seeded into T175 tissue cultureflasks at 5 million cells per flask for expansion in monolayer andcultured in DMEM supplemented with 10% calf serum and 1x PSG. Cells areincubated at 37° C. with humidified air containing 5% CO₂. The culturemedium is changed every 4 days. Human chondrocytes are ready to be used(e.g. bioprinted) when 80% to 90% confluence is reached (1 to 2 weeks inprimary culture). All cells used for bioprinting are first or secondpassage.

During the surgery to repair the chondral defect, a bioprinter isattached to a robotic arm. The bioprinter comprises a syringe. Thesyringe contains a bio-ink comprising the individual’s previouslyharvested chondrocytes and a hydrogel such as Matrigel®. An endoscope isfurther placed near the defect in order to monitor, in real time, thebioprinting process, with the surgeon manually adjusting any bioprintingparameter as needed. The surgeon positions the bioprinter, and thenengages the control system to begin bioprinting. The bio-rink comprisingthe chondrocytes and Matrigel® is extruded onto the chondral defect.

Example 2: Repairing an Osteochondral Defect in the Knee During Surgery,Using Four Printheads in a Single Bioprinter

During a surgery to repair an osteochondral defect in an individual, abioprinter is attached to a robotic arm. The bioprinter comprises fourprintheads. The first printhead comprises the individual’s previouslyharvested chondrocytes. The second printhead comprises the individual’spreviously harvested osteoblasts. The third printhead comprisesalginate. The fourth printhead comprises calcium chloride. Allprintheads utilize ink-jet based printing and comprise 300 printnozzles, set at a distance of 1 to 2 mm from the osteochondral defect. Acombination of the osteoblasts, alginate, and calcium chloride isprinted onto the bone defect of the osteochondral defect. A combinationof the chondrocytes, alginate, and calcium chloride is then printed ontothe cartilage defect of the osteochondral defect.

An endoscope is further placed near the osteochondral defect in order tomonitor, in real time, the bioprinting process, with the surgeonmanually adjusting any bioprinting parameter as needed. The surgeonpositions the bioprinter, and then engages the control system to beginbioprinting.

Example 3: Repairing a Chondral Defect in Knee Cartilage During Surgery,Using UV Cross Linking

During a surgery to repair a chondral defect in an individual, abioprinter comprising a printhead is attached to a robotic arm. PurifiedPEGDMA is dissolved in PBD to a concentration of 10% w/v. Photoinitiator1-2959 (Irgacure®) is added at a final concentration of 0.05 w/v. Thebio-ink is made up of the individual’s previously harvested chondrocytessuspended in filter-sterilized PEGDMA solution at 5 × 10⁶ cells/mL. A UVlamp is attached to an endoscope.

The printhead is set at a distance of 1 to 2 mm from the defect. Theendoscope is placed near the defect in order to monitor, in real time,the bioprinting process, with the surgeon manually adjusting anybioprinting parameter as needed. The surgeon positions the bioprinter,and then engages the control system to begin bioprinting. As the bio-inkit being ejected, it is exposed to UV light emitted from the UV lamp ata wavelength of 280 nm, thereby cross-linking the PEDGMA.

Example 4: Repairing an Osteochondral Defect in the Knee During Surgery,Using UV Crosslinking

During a surgery to repair an osteochondral defect in an individual, abioprinter comprising two printheads is attached to a robotic arm. Theprintheads of the bioprinter comprises 300 print nozzles, set at adistance of 1 to 2 mm from the defect. The first printhead contains abio-ink comprising an individual’s previously harvested chondrocytes, amethacrylated hyaluronic acid (MeHA), and the photoinitiator 1-2959(Irgacure®). The second printhead contains a bio-ink comprising anindividual’s previously harvested osteoblasts, a methacrylatedhyaluronic acid (MeHA), and the photoinitiator 1-2959 (Irgacure®). Anendoscope is placed near the osteochondral defect in order to monitor,in real time, the bioprinting process, with the surgeon manuallyadjusting any bioprinting parameter as needed. The surgeon positions thebioprinter, and then engages the control system to begin bioprinting.Several layers of the bio-ink comprising the osteoblasts are applied tothe osteochondral defect first, followed by several layers of thebio-ink comprising the chondrocytes. A UV lamp attached to the endoscopeemits UV light at 280 nm onto the area while the bio-ink construct isbeing printed, thereby crosslinking the construct.

Example 5: Repairing an Osteochondral Defect in the Knee During Surgery,Using a Robotic Arm Controlled by a Surgeon

During a surgery to repair an osteochondral defect in an individual, abioprinter comprising printheads, is attached to a robotic arm. Anendoscope is placed near the osteochondral defect in order to monitor,in real time, the bioprinting process, with the surgeon manuallyadjusting any bioprinting parameter as needed. The surgeon positions thebioprinter, and then engages the control system to begin bioprinting.The robotic arm only prints when the printhead is pointing at the targetand turns off if moved away from the target area. Several layers of thebio-ink comprising a plurality of osteoblasts is applied to theosteochondral defect first, followed by several layers of the bio-inkcomprising a plurality of chondrocytes. The control system turns on theprinthead with the appropriate cell type when printing each layer (i.e.the control system turns on a first printhead loaded with the bio-inkcomprising osteoblasts only when printing the bone layer and the controlsystem turns on a second printhead loaded with the bio-ink comprisingchondrocytes only when printing the cartilage layer). A UV lamp attachedto the endoscope emits UV light at 280 nm onto the area while thebio-ink construct is being printed, thereby crosslinking the construct.Having the surgeon control the motion of the robotic arm increasessafety and reduces the need for a mechanical actuation of the roboticarm. The robotic control of the printheads ensures that cells are onlyprinted at the desired location.

Example 6: Repairing an Osteochondral Defect in the Knee During Surgery,Using a Robotic Arm Controlled by a Computer

During a surgery to repair an osteochondral defect in an individual, abioprinter comprising printheads is attached to a robotic arm. Anendoscope is placed near the osteochondral defect in order to monitor,in real time, the bioprinting process, with the surgeon manuallyadjusting any bioprinting parameter as needed. The surgeon positions thebioprinter, and then engages the control system to begin bioprinting.The control system controls the motion of the robotic arm throughactuators and only prints when the printhead is pointing at the target.Several layers of the bio-ink comprising the osteoblasts are applied tothe osteochondral defect first, followed by several layers of thebio-ink comprising the chondrocytes. The control system turns on theprinthead with the appropriate cell type when printing each layer (i.e.the control system turns on a first printhead loaded with the bio-inkcomprising osteoblasts only when printing the bone layer and the controlsystem turns on a second printhead loaded with the bio-ink comprisingchondrocytes only when printing the cartilage layer). A computerizedvisual feedback system provides real time imaging of the defect beingprinted and controls the motion of the robotic arm and the firing of theprintheads to account for errors in printing or missed print areas. A UVlamp attached to the endoscope emits UV light at 280 nm onto the areawhile the bio-ink construct is being printed, thereby crosslinking theconstruct. Having the control system control the motion of the roboticarm increases accuracy when following a preoperative plan. The roboticcontrol of the printheads ensures that cells are only printed at thedesired location.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1-31. (canceled)
 32. A method of bioprinting a bio-ink construct on aninternal tissue defect during a minimally invasive surgery on anindividual in need thereof, comprising: a. visualizing the internaltissue defect; b. positioning a bioprinter comprising a printhead withinproximity of or in contact with the internal tissue defect; and c.ejecting a bio-ink from the printhead onto the internal tissue defect toform a bio-ink layer, thereby generating a bio-ink construct.
 33. Themethod of claim 32, wherein the bio-ink construct comprises a pluralityof bio-ink layers.
 34. The method of claim 32, wherein the bio-inkconstruct is a live tissue.
 35. The method of claim 32, wherein theprinthead comprises a needle, an extended cylinder, a fluid line, aprint nozzle, or a plurality of print nozzles.
 36. The method of claim32, further comprising polymerizing the bio-ink.
 37. The method of claim32, wherein the bioprinter further comprises a second printhead.
 38. Themethod of claim 32, further comprising positioning a second bioprintercomprising a second printhead within proximity of or in contact with theinternal tissue defect.
 39. The method of claim 32, further comprisingejecting a second bio-ink from the second printhead of the secondbioprinter onto the internal tissue defect to form a second bio-inklayer.
 40. The method of claim 32, further comprising controlling thebioprinter with a control system.
 41. The method of claim 40, whereinthe control system controls a bio-ink printing parameter.
 42. The methodof claim 41, wherein the bio-ink printing parameter comprisestemperature, back-pressure, drops per nozzle, frequency of drop rate,number of nozzles in use, firing energy, resolution, viscosity, cellconcentration, physiological temperature, speed of printing, or acombination thereof.
 43. The method of claim 40, wherein the methodfurther comprises controlling a robotic arm operatively connected to thecontrol system.
 44. The method of claim 43, wherein the method comprisescontrolling the robotic arm to perform at least one of: 1) positioningthe bioprinter within proximity of or in contact with the internaltissue defect, and 2) moving the bioprinter with six degrees of freedom.45. The method of claim 35, wherein the method further comprisescontrolling and actuating a first print nozzle of the plurality of printnozzles independently of controlling and actuating a second print nozzleof the plurality of print nozzles.
 46. The method of claim 32, whereinthe printhead ejects the bio-ink continuously.
 47. The method of claim32, further comprising positioning an endoscope within proximity of theinternal tissue defect.
 48. The method of claim 47, wherein theendoscope visualizes the internal tissue defect.
 49. The method of claim32, wherein the bio-ink comprises a plurality of cells, a component ofextracellular matrix, a synthetic polymer, a natural polymer, across-linking agent, a photoinitiator, or a combination thereof.
 50. Themethod of claim 32, wherein the internal tissue defect comprises achondral defect.
 51. The method of claim 50, wherein the chondral defectcomprises an osteochondral defect.